Conformational probes and methods for sequencing nucleic acids

ABSTRACT

This disclosure provides a method of determining a sequence of nucleotides for a nucleic acid template. The method can include the steps of contacting the nucleic acid template with a conformationally labeled polymerase and at least four different nucleotide species under conditions wherein the conformationally labeled polymerase catalyzes sequential addition of the nucleotide species to form a nucleic acid complement of the nucleic acid template, wherein the sequential addition of each different nucleotide species produces a conformational signal change from the conformationally labeled polymerase and wherein the rate or time duration for the conformational signal change is distinguishable for each different nucleotide species; detecting a series of changes in the signal from the conformationally labeled polymerase under the conditions; and determining the rates or time durations for the changes in the signal, thereby determining the sequence of nucleotides for the nucleic acid template.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of, and claims the benefits andpriority to, U.S. patent application Ser. No. 17/091,845, filed Nov. 6,2020, which is a continuation of U.S. patent application Ser. No.16/352,731, filed Mar. 13, 2019 (now U.S. Pat. No. 10,837,056), which isa continuation of U.S. patent application Ser. No. 15/853,118, filedDec. 22, 2017 (now U.S. Pat. No. 10,233,493), which is a continuation ofU.S. application Ser. No. 15/132,662, filed Apr. 19, 2016 (now U.S. Pat.No. 9,862,998), which is a continuation of U.S. application Ser. No.13/162,325, filed Jun. 16, 2011 (now U.S. Pat. No. 9,353,412), whichclaims priority to U.S. Provisional Application Ser. No. 61/356,178,filed Jun. 18, 2010; 61/433,025 filed Jan. 14, 2011, and 61/437,441,filed Jan. 28, 2011, each of which is incorporated herein by referencein its entirety.

BACKGROUND

This disclosure relates generally to evaluation of nucleic acids andenzymes that catalyze reactions having nucleic acids as their reactantsor products. More specifically this disclosure relates to sequencingnucleic acids, evaluating activity of polymerases or other enzymes, orcombinations thereof.

Our genome provides a blue print with boundless potential for predictingmany of our inherent predispositions such as our likes, dislikes,talents, emotional inclinations and susceptibility to disease. Ourability to decipher the blue print is slowly improving throughimprovements in nucleic acid sequencing technologies. However, to dateonly a handful of human genomes have been sequenced. Having one or even100 genome sequences is scientifically interesting because it providesclues to unraveling the symbols and features that make up the blueprint. However, a more complete understanding of how the information ineach blue print relates to the living structures they encode, willrequire that tens-of-thousands or millions of genomes be sequenced. Onlythen will scientists be able to correlate the complexities of thegenetic code with the variety of human characteristics.

With sufficient numbers of genomic sequences of many different people,researchers will be able to identify the appropriate correlations to (a)guide individuals in making proper choices for preventive medicine, (b)develop targeted drugs and other treatments that are specific andeffective, and avoid side effects and drug resistance, and (c) reducethe costs to society and the individual in implementing effectivetherapies based on an individual's genomic predispositions.

The day when each person can sit down with a doctor to review a copy oftheir own personal genome and determine appropriate choices for ahealthy lifestyle or a proper course of treatment for a presentingdisease is not here yet. First the time and cost for determining genomicsequences must come down to a level that large genetic correlationstudies can be carried out by scientists. Furthermore, the technologymust reach the point that it is accessible to virtually anyone in aclinical environment regardless of economic means and personalsituation.

Thus, there exists a need for improved nucleic acid sequencingtechniques. The present invention satisfies this need and provides otheradvantages as well.

BRIEF SUMMARY

This disclosure provides a method of determining a sequence ofnucleotides for a nucleic acid template. The method can include thesteps of contacting the nucleic acid template with a conformationallylabeled polymerase and at least four different nucleotide species underconditions wherein the conformationally labeled polymerase catalyzessequential addition of the nucleotide species to form a nucleic acidcomplement of the nucleic acid template, wherein the sequential additionof each different nucleotide species produces a conformational signalchange from the conformationally labeled polymerase and wherein the rateor time duration for the conformational signal change is distinguishablefor each different nucleotide species; detecting a series of changes inthe signal from the conformationally labeled polymerase under theconditions; and determining the rates or time durations for the changesin the signal, thereby determining the sequence of nucleotides for thenucleic acid template. The method can also be used to identify modifiedbases within a sequence such as methylated bases.

Also provided herein is a method of determining a sequence ofnucleotides for a nucleic acid template. The method can include thesteps of contacting the nucleic acid template with a conformationallylabeled exonuclease under conditions wherein the conformationallylabeled exonuclease catalyzes sequential removal of nucleotide speciesfrom the nucleic acid template, wherein the sequential removal of eachdifferent nucleotide species produces a conformational signal changefrom the conformationally labeled exonuclease and wherein the rate ortime duration for the conformational signal change is distinguishablefor each different nucleotide species that is removed; detecting aseries of changes in the signal from the conformationally labeledexonuclease under the conditions; and determining the rates or timedurations for the changes in the signal for the series of changes in thesignal from the conformationally labeled exonuclease, therebydetermining the sequence of nucleotides for the nucleic acid template.The method can also be used to identify modified bases within a sequencesuch as methylated bases.

This disclosure further provides a method of determining a sequence ofnucleotides for a nucleic acid template. The method can include thesteps of contacting the nucleic acid template with a conformationallylabeled polymerase and at least four different nucleotide species underconditions wherein the conformationally labeled polymerase catalyzessequential addition of the nucleotide species to form a nucleic acidcomplement of the nucleic acid template, wherein the sequential additionof each different nucleotide species produces a conformational signalchange from the conformationally labeled polymerase and wherein the rateor time duration for the conformational signal change is distinguishablefor each different nucleotide species; detecting a series of changes inthe signal from the conformationally labeled polymerase under theconditions; determining the rates or time durations for the changes inthe signal, thereby determining the sequence of nucleotides for thenucleic acid template; contacting the nucleic acid template with aconformationally labeled exonuclease under conditions wherein theconformationally labeled exonuclease catalyzes sequential removal ofnucleotide species from the nucleic acid template, wherein thesequential removal of each different nucleotide species produces aconformational signal change from the conformationally labeledexonuclease and wherein the rate or time duration for the conformationalsignal change is distinguishable for each different nucleotide speciesthat is removed; detecting a series of changes in the signal from theconformationally labeled exonuclease under the conditions; anddetermining the rate or time durations for the changes in the signal forthe series of changes in the signal from the conformationally labeledexonuclease. The method can also be used to identify modified baseswithin a sequence such as methylated bases.

In a further embodiment a method of determining a sequence ofnucleotides for a nucleic acid sample is provided. The method caninclude the steps of providing an array of nucleic acid templates,wherein the nucleic acid templates include nucleotide sequence fragmentsof the nucleic acid sample; contacting the array of nucleic acidtemplates with conformationally labeled polymerases and at least fourdifferent nucleotide species under conditions wherein theconformationally labeled polymerases catalyze sequential addition of thenucleotide species to form nucleic acid complements of the nucleic acidtemplates, wherein the sequential addition of each different nucleotidespecies produces a conformational signal change from theconformationally labeled polymerase and wherein the rate or timeduration for the conformational signal change is distinguishable foreach different nucleotide species; detecting a series of changes in thesignal from the conformationally labeled polymerase under the conditionsand at individual locations of the array; and determining the rates ortime durations for the changes in the signal at the individual locationsof the array, thereby determining the sequence of nucleotides for thenucleic acid sample. The method can also be used to identify modifiedbases within a sequence such as methylated bases.

Also provided is a method of determining nucleotide sequences which canoptionally include the steps of (a) providing an array of nucleic acidtemplates; (b) providing a mixture of nucleotide species, the mixtureincluding (i) at least four different nucleotide species, (ii) at leastone of the four different nucleotide species having a reversibleterminator moiety, and (iii) at least two of the four differentnucleotide species having an extendible 3′ hydroxyl moiety; (c)contacting the array of nucleic acid templates with conformationallylabeled polymerases and the mixture of nucleotide species underconditions wherein the conformationally labeled polymerases catalyzesequential addition of the nucleotide species to form nucleic acidcomplements of the nucleic acid templates, wherein the sequentialaddition of each different nucleotide species produces a conformationalsignal change from the conformationally labeled polymerase, wherein therate or time duration for the conformational signal change isdistinguishable for the at least two nucleotide species having theextendible 3′ hydroxyl moiety, and wherein a plurality of the nucleicacid complements incorporate the at least one nucleotide species thathas the reversible terminator moiety; (d) removing the reversibleterminator moiety; (e) detecting a series of changes in the signal fromthe conformationally labeled polymerase at individual locations of thearray; and (0 determining the sequence of nucleotides for the nucleicacid sample from the series of changes in the signal from theconformationally labeled polymerase.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a molecular model of Phi29 polymerase.

FIG. 2 shows a kinetic model of polymerase activity.

FIG. 3A-D shows triphosphates having chemical modifications at variouspositions.

FIG. 4 shows exemplary moieties that can be attached to a nucleotide viaa gamma-phosphoamidite linker.

FIG. 5 shows a plot of donor fluorescence vs. time for incorporation ofnatural nucleotides dATP, dGTP, dTTP and dCTP into a template boundprimer by a conformationally labeled polymerase.

FIG. 6A-C shows a plot of donor fluorescence vs. time for incorporationof nucleotides 1-alpha-bromo-dCTP (panel A), 1-alpha-thiol-dCTP (panelB) and dCTP (panel C) into a template bound primer by a conformationallylabeled polymerase.

FIG. 7 shows a plot of donor fluorescence vs. time for incorporation ofnucleotides dTTP and dUTP into a template bound primer by aconformationally labeled polymerase.

FIG. 8 shows a plot of donor fluorescence vs. time for incorporation ofcorrect and incorrect nucleotides into a template bound primer by aconformationally labeled polymerase.

FIG. 9A-B shows a plot of 5-TAMRA fluorescence vs. time forincorporation of correct nucleotide (panel A) and incorrect nucleotides(panel B) into a template bound primer by a conformationally labeledpolymerase.

FIG. 10A-D shows a plot of 5-TAMRA fluorescence vs. time forincorporation of natural nucleotides into a template bound primer by aconformationally labeled polymerase.

FIG. 11A-B shows a plot of 5-TAMRA fluorescence vs. time forincorporation of non-natural nucleotides into a template bound primer bya conformationally labeled polymerase.

FIG. 12 shows a plot of donor fluorescence vs. time for incorporation ofdTTP and γ-dTTP into a template bound primer by a conformationallylabeled polymerase.

FIG. 13A-B shows a plot of 5-TAMRA fluorescence vs. time forincorporation of dTTP (Panel A) and γ-dTTP (Panel B) into a templatebound primer by a conformationally labeled polymerase.

FIG. 14A-B shows a method for light-gated sequencing.

FIG. 15 shows an absorption spectrum for a conformationally labeledpolymerase. The absorption peaks for the polymerase, Cy3 and AF647 aredemarcated.

FIG. 16 shows a plot of fluorescence quenching vs. time for a polymeraseextension reaction

DETAILED DESCRIPTION OF EMBODIMENTS

This disclosure provides methods useful for determining the nucleotidesequences of nucleic acid molecules. The methods can be used to identifythe nucleotide sequence for a single species of nucleic acid, forexample, present in a reaction vessel or attached to a solid support.For purposes of illustration, the methods will often be exemplified inthe context of steps carried out for a single nucleic acid molecule orsingle species of nucleic acid molecule. However, the methods set forthherein are useful for multiplex detection whereby the steps are carriedout simultaneously for several different nucleic acids. The methods canbe carried out at a multiplex level that allows a substantial portion,or in some cases an entire genome, to be sequenced simultaneously.Furthermore, whether or not multiplex detection is carried out,detection can occur at a single molecule detection level or at a levelwhereby several species of a particular molecule are detected as anensemble.

In particular embodiments, a sequence of nucleotides for a nucleic acidtemplate can be determined based on conformational changes occurring inan enzyme that interacts with the nucleic acid. Such enzymes, oftenreferred to as nucleic acid enzymes, can interact sequentially with thenucleotide subunits of a nucleic acid in order to carry out a series ofreactions on the nucleic acid. Distinguishing the conformational changesthat occur for each type of nucleotide that the enzyme interacts withand determining the sequence of those changes can be used to determinethe sequence of the nucleic acid. For example, a polymerase can use afirst nucleic acid strand as a template to sequentially build a second,complementary nucleic acid strand by sequential addition of nucleotidesto the second strand. The polymerase undergoes conformational changeswith each nucleotide addition. As set forth in further detail herein,the conformational changes that occur for each type of nucleotide thatis added can be distinguished and the sequence of those changes can bedetected to determine the sequence of either or both of the nucleic acidstrands. In another example, an exonuclease can sequentially removenucleotides from a nucleic acid. Conformational changes that occur foreach type of nucleotide that is removed can be distinguished and thesequence of those changes can be detected to determine the sequence ofthe nucleic acid.

Also provided herein are compositions useful for determining thenucleotide sequences of nucleic acid molecules. An example is a nucleicacid enzyme that is labeled to produce one or more signals indicative ofa conformational change in the enzyme as it interacts with one or morereactants such as a nucleic acid or nucleotide. For example, apolymerase can be conformationally labeled to allow detection of asignal indicative of nucleotide binding, a signal indicative of additionof a nucleotide to a growing nucleic acid molecule, or a signalindicative of an intermediate change in the conformation of thepolymerase between binding and catalysis. In particular embodiments, thesignal can distinguish a binding event from a catalytic event. However,such a distinction may not be necessary for some embodiments and thesignal can be merely indicative of the overall addition of a nucleotide.Alternatively or additionally, the signal can distinguish binding of acorrectly base-paired nucleotide from binding of an incorrectlybase-paired nucleotide. Another example is an exonuclease that isconformationally labeled to allow detection of a signal indicative ofcatalytic breakage of a bond between a nucleotide and nucleic acid, asignal indicative of dissociation of the nucleotide from theexonuclease, or a signal indicative of an intermediate change in theconformation of the exonuclease between catalysis and dissociation. Theexonuclease can be a polymerase acting under conditions to removenucleotides from a nucleic acid, for example, via 3′→5′ exonucleolyticcleavage activity. Again, in particular embodiments the catalytic eventcan be distinguished from the dissociation event, or the signal can beindicative of the overall removal of a nucleotide.

Another example of a useful type of composition is a nucleotide analogthat is incorporated into a polynucleotide strand by a polymerase at arate that is measurably different than the rate at which anothernucleotide is incorporated into the strand by the polymerase. Anotheruseful nucleotide analog is one that is bound to a polymerase at a ratethat is measurably different than the rate at which another nucleotideis bound to the polymerase. A nucleotide analog that causes aconformational change of a polymerase at a rate that is measurablydifferent than for another nucleotide is also useful. The relative rateof binding, incorporation or polymerase conformational change for anucleotide analog can be measured relative to a natural nucleotidehaving the same Watson-Crick base pairing partner or relative to othernucleotides that are used in a nucleic acid synthesis reaction. Therelative rate can be faster or slower for the nucleotide analog. Anucleotide analog that is removed from a nucleic acid by an exonucleaseat a rate that is measurably faster or slower than the rate at which anatural nucleotide is removed from the nucleic acid is also useful.Another useful nucleotide analog is one that causes conformationalchange of an exonuclease at a rate that is measurably different than therate for another nucleotide.

For purposes of demonstration several compositions, such asconformationally labeled molecules and nucleotide analogs, are describedin the context of particular nucleic acid sequencing methods. It will beunderstood that the compositions set forth herein can be used in avariety of other sequencing methods. Moreover, the compositions can beused for any of a variety of applications such as those that will beapparent to those skilled in the art in view of the known ordeterminable properties of the compositions and the guidance set forthherein.

Terms used herein will be understood to take on their ordinary meaningunless specified otherwise. Examples of several terms used herein andtheir definitions are set forth below.

As used herein, the term “conformationally labeled,” when used inreference to a molecule, means having at least one probe that isresponsive to a change in the structure of the molecule, a change in theshape of the molecule or a change in the arrangement of parts of themolecule. The molecule can be, for example, a polymerase, reversetranscriptase, exonuclease or other nucleic acid enzyme such as thoseset forth herein below. The parts of the molecule can be, for example,atoms that change relative location due to rotation about one or morechemical bonds occurring in the molecular structure between the atoms.The parts of the molecule can be domains of a macromolecule such asthose commonly known in the relevant art. For example, polymerasesinclude domains referred to as the finger, palm and thumb domains. Inthe case of proteins the parts can be regions of secondary, tertiary orquaternary structure. The probe(s) can be attached to the molecule, forexample, via a covalent linkage. However, the probe(s) need not beattached to the molecule, being, for example, located in proximity tothe molecule. In particular embodiments the probe is not attached to areactant or product of the molecule such as a nucleotide or nucleicacid.

As used herein, the term “conformational signal change,” when used inreference to a conformationally labeled molecule, means the appearance,disappearance, or alteration of a detectable signal from a probe of themolecule in response to a change in the structure, shape or arrangementof parts of the molecule. For example, the signal change can be due to achange in the interaction of the probe with a first portion of themolecule to interact with a second portion of the molecule. The term,when specifically recited, is intended to distinguish from changes insignal that arise from a probe of a molecule due to a change in theinteraction of the probe with a reactant that binds specifically to themolecule or a change in the interaction of the probe with a product thatresults from catalytic activity of the molecule. For example, the term,when specifically recited, can be used to exclude a change in afluorescence resonance energy transfer signal that arises from a donoror acceptor probe of a polymerase changing its interaction with anacceptor or donor probe, respectively, on a nucleic acid or nucleotide.Also by way of example, the term, when specifically recited, can be usedto exclude a change in a fluorescence signal that arises from a probe ona polymerase or quencher on a polymerase that changes its interactionwith a quencher or probe, respectively, on a nucleic acid or nucleotide.

As used herein, the term “position,” when used in reference to aprotein, means a location for a particular amino acid residue in thestructure of the protein. The term can be used to describe the locationindependent of the type of amino acid residue that is present at theposition. Thus, the position can be occupied by an amino acid residuethat is found in a wild-type protein or the position can be occupied byanother amino acid residue due to a mutation at the position.Furthermore, the term can be used to describe a location that ishomologous across two or more proteins of the same type. Homologouslocations are known or identifiable to those in the art based onstructural comparison between proteins of the same type. For example,position 486 in Pfu DNA polymerase is homologous to 485 in 9° N DNApolymerase, position 488 in Vent DNA polymerase, and position 485 inJDF-3 DNA polymerase. The term “position” can also be used in referenceto a nucleic acid in order to identify the location for a particularnucleotide in the sequence of the nucleic acid. The location can beidentified independent of the type of nucleotide that is present at theposition.

As used herein, the term “species” is used to identify molecules thatshare the same chemical structure. For example, a mixture of nucleotidescan include several dCTP molecules. The dCTP molecules will beunderstood to be the same species as each other. Similarly, individualDNA molecules that have the same sequence of nucleotides are the samespecies.

As used herein, the term “complement” or grammatical variations thereofcan be used to describe the characteristic of a nucleic acid whereby itsbases precisely pair with those of a second nucleic acid. For twonucleic acid strands that are complementary the sequence of bases forone strand can be used to determine the sequence of bases for the other.Complementarity includes for example base pairing between naturallyoccurring bases such as the pairing of cytosine with guanine and thepairing of adenine with thymine. However, complementarity can also occurbetween nucleotide analogs having non-natural bases. For example, anucleic acid strand having non-natural bases can form a complement to asecond strand having natural bases such that the sequence of bases forthe former can be used to determine the nucleotide sequence for thelatter and vice versa. Complementarity can be, but need not necessarilybe, perfect for particular embodiments set forth herein. For example,two nucleic acids can have at least about 75% complementarity, at leastabout 80% complementarity, at least about 85% complementarity, at leastabout 90% complementarity, at least about 95% complementarity, at leastabout 98% complementarity, or 100% complementarity.

As used herein, the term “array” refers to a population of differentmolecules that are attached to one or more solid-phase substrates suchthat the different molecules can be differentiated from each otheraccording to their relative location. An array can include differentmolecules that are each located at a different addressable location on asolid-phase substrate. Alternatively, an array can include separatesolid-phase substrates each bearing a different molecule, wherein thedifferent probe molecules can be identified according to the locationsof the solid-phase substrates on a surface to which the solid-phasesubstrates are attached or according to the locations of the solid-phasesubstrates in a liquid such as a fluid stream. The molecules of thearray can be nucleic acid primers, nucleic acid probes, nucleic acidtemplates or nucleic acid enzymes such as polymerases and exonucleases.

As used herein, the term “nucleic acid fragment” includes a nucleic acidthat has a portion of contiguous sequence from a larger nucleic acid.The fragment can be a piece that is removed from a larger molecule, forexample, by physical shearing, chemical cleavage or enzymatic (nuclease)cleavage. A fragment can also be a product of amplifying a region orportion of a larger sequence, for example, using PCR primers thathybridize to sites within a chromosome such that a region internal tothe chromosome is amplified and flanking regions are not. A fragment canalso be a product of a transposase reaction such as a reaction describedin Adey et al. Genome Biology 11:R119 (2010) and U.S. Pat. No. 5,965,443or 6,437,109, each of which is incorporated herein by reference.

As used herein, the term “nucleotide” is intended to include naturalnucleotides, analogs thereof, ribonucleotides, deoxyribonucleotides,dideoxyribonucleotides and other molecules known as nucleotides. Theterm can be used to refer to a monomeric unit that is present in apolymer, for example to identify a subunit present in a DNA or RNAstrand. The term can also be used to refer to a molecule that is notnecessarily present in a polymer, for example, a molecule that iscapable of being incorporated into a polynucleotide in a templatedependent manner by a polymerase. The term can refer to a nucleosideunit having, for example, 0, 1, 2, 3 or more phosphates on the 5′carbon. For example, tetraphosphate nucleotides and pentaphosphatenucleotides can be particularly useful. Exemplary natural nucleotidesinclude, without limitation, ATP, UTP, CTP, GTP, ADP, UDP, CDP, GDP,AMP, UMP, CMP, GMP, dATP, dTTP, dCTP, dGTP, dADP, dTDP, dCDP, dGDP,dAMP, dTMP, dCMP, and dGMP.

Non-natural nucleotides also referred to herein as nucleotide analogs,include those that are not present in a natural biological system or notsubstantially incorporated into polynucleotides by a polymerase in itsnatural milieu, for example, in a non-recombinant cell that expressesthe polymerase. Particularly useful non-natural nucleotides includethose that are incorporated into a polynucleotide strand by a polymeraseat a rate that is substantially faster or slower than the rate at whichanother nucleotide, such as a natural nucleotide that base-pairs withthe same Watson-Crick complementary base, is incorporated into thestrand by the polymerase. For example, a non-natural nucleotide may beincorporated at a rate that is at least 2 fold different, 5 folddifferent, 10 fold different, 25 fold different, 50 fold different, 100fold different, 1000 fold different, 10000 fold different or more whencompared to the incorporation rate of a natural nucleotide, such as oneor more of those exemplified above. A non-natural nucleotide can becapable of being further extended after being incorporated into apolynucleotide. Examples include, nucleotide analogs having a 3′hydroxyl or nucleotide analogs having a reversible blocking group at the3′ position that can be removed to allow further extension of apolynucleotide that has incorporated the nucleotide analog. Examples ofreversible blocking groups that can be used are described, for example,in U.S. Pat. Nos. 7,427,673; 7,414,116; and 7,057,026 and PCTpublications WO 91/06678 and WO 07/123744, each of which is incorporatedherein by reference. It will be understood that in some embodiments anucleotide analog having a 3′ blocking group or lacking a 3′ hydroxyl(such as a dideoxynucleotide analog) can be used under conditions wherethe polynucleotide that has incorporated the nucleotide analog is notfurther extended. In some embodiments, the nucleotide(s) will notinclude a reversible blocking group, or the nucleotides(s) will notinclude a non-reversible blocking group or the nucleotide(s) will notinclude any blocking group at all.

Provided herein is a method of determining a sequence of nucleotides fora nucleic acid template. The method can include the steps of contactingthe nucleic acid template with a conformationally labeled polymerase andat least four different nucleotide species under conditions wherein theconformationally labeled polymerase catalyzes sequential addition of thenucleotide species to form a nucleic acid complement of the nucleic acidtemplate, wherein the sequential addition of each different nucleotidespecies produces a conformational signal change from theconformationally labeled polymerase and wherein the rate or timeduration for the conformational signal change is distinguishable foreach different nucleotide species; detecting a series of changes in thesignal from the conformationally labeled polymerase under theconditions; and determining the rates or time durations for the changesin the signal, thereby determining the sequence of nucleotides for thenucleic acid template.

Any of a variety of polymerases can be used in a method or compositionset forth herein including, for example, protein-based enzymes isolatedfrom biological systems and functional variants thereof. Reference to aparticular polymerase, such as those exemplified below, will beunderstood to include functional variants thereof unless indicatedotherwise. A particularly useful function of a polymerase is to catalyzethe polymerization of a nucleic acid strand using an existing nucleicacid as a template. Other functions that are useful are describedelsewhere herein. Examples of useful polymerases include DNA polymerasesand RNA polymerases. Exemplary DNA polymerases include those that havebeen classified by structural homology into families identified as A, B,C, D, X, Y, and RT. DNA Polymerases in Family A include, for example, T7DNA polymerase, eukaryotic mitochondrial DNA Polymerase γ, E. coli DNAPol I, Thermus aquaticus Pol I, and Bacillus stearothermophilus Pol I.DNA Polymerases in Family B include, for example, eukaryotic DNApolymerases a, 8, and c; DNA polymerase ζ; T4 DNA polymerase, Phi29 DNApolymerase, and RB69 bacteriophage DNA polymerase. Family C includes,for example, the E. coli DNA Polymerase III alpha subunit. Family Dincludes, for example, polymerases derived from the Euryarchaeotasubdomain of Archaea. DNA Polymerases in Family X include, for example,eukaryotic polymerases Pol β, pol σ, Pol λ, and Pol μ, and S. cerevisiaePol4. DNA Polymerases in Family Y include, for example, Pol μ, Pol iota,Pol kappa, E. coli Pol IV (DINB) and E. coli Pol V (UmuD′2C). The RT(reverse transcriptase) family of DNA polymerases includes, for example,retrovirus reverse transcriptases and eukaryotic telomerases. ExemplaryRNA polymerases include, but are not limited to, viral RNA polymerasessuch as T7 RNA polymerase; Eukaryotic RNA polymerases such as RNApolymerase I, RNA polymerase II, RNA polymerase III, RNA polymerase IV,and RNA polymerase V; and Archaea RNA polymerase.

The above classifications are provided for illustrative purposes. Itwill be understood that variations in the classification system arepossible. For example, in at least one classification system Family Cpolymerases have been categorized as a subcategory of Family X.Furthermore, polymerases can be classified according to othercharacteristics, whether functional or structural, that may or may notoverlap with the structural characteristics exemplified above. Someexemplary characteristics are set forth in further detail below.

A polymerase having an intrinsic 3′-5′ proofreading exonuclease activitycan be useful for some embodiments. Polymerases that substantially lack3′-5′ proofreading exonuclease activity are also useful in someembodiments, for example, in most sequencing embodiments. Absence ofexonuclease activity can be a wild type characteristic or acharacteristic imparted by a variant or engineered polymerase structure.For example, exo minus Klenow fragment is a mutated version of Klenowfragment that lacks 3′-5′ proofreading exonuclease activity. Klenowfragment and its exo minus variant can be useful in a method orcomposition set forth herein. Polymerases can also catalyzepyrophosphorolysis, the direct reversal of polymerization in the sameactive site. This activity can be useful for various embodiments thatare set forth herein.

Polymerases can be characterized according to their processivity. Apolymerase can have an average processivity that is at least about 50nucleotides, 100 nucleotides, 1,000 nucleotides, 10,000 nucleotides,100,000 nucleotides or more. Alternatively or additionally, the averageprocessivity for a polymerase used as set forth herein can be, forexample, at most 1 million nucleotides, 100,000 nucleotides, 10,000nucleotides, 1,000 nucleotides, 100 nucleotides or 50 nucleotides.Polymerases can also be characterized according to their rate ofprocessivity or nucleotide incorporation. For example, many nativepolymerases can incorporate nucleotides at a rate of at least 1,000nucleotides per second. In some embodiments a slower rate may bedesired. For example, an appropriate polymerase and reaction conditionscan be used to achieve an average rate of at most 500 nucleotides persecond, 100 nucleotides per second, 10 nucleotides per second, 1nucleotide per second, 1 nucleotide per 10 seconds, 1 nucleotide perminute or slower. As set forth in further detail elsewhere herein,nucleotide analogs can be used that have slower or faster rates ofincorporation than naturally occurring nucleotides. It will beunderstood that polymerases from any of a variety of sources can bemodified to increase or decrease their average processivity or theiraverage rate of processivity (e.g. average rate of nucleotideincorporation) or both. Accordingly, a desired reaction rate can beachieved using appropriate polymerase(s), nucleotide analog(s), nucleicacid template(s) and other reaction conditions.

Depending on the embodiment that is to be used, a polymerase can beeither thermophilic or heat inactivatable. Thermophilic polymerases aretypically useful for high temperature conditions or in thermocyclingconditions such as those employed for polymerase chain reaction (PCR)techniques. Examples of thermophilic polymerases include, but are notlimited to 9° N DNA Polymerase, Taq DNA polymerase, Phusion® DNApolymerase, Pfu DNA polymerase, RB69 DNA polymerase, KOD DNA polymerase,and VentR® DNA polymerase. Most polymerases isolated fromnon-thermophilic organisms are heat inactivatable. Examples are DNApolymerases from phage. It will be understood that polymerases from anyof a variety of sources can be modified to increase or decrease theirtolerance to high temperature conditions.

Polymerases can be characterized according to their fidelity. Fidelitygenerally refers to the accuracy with which a polymerase incorporatescorrect nucleotides into a copy of a nucleic acid template. DNApolymerase fidelity can be measured as the ratio of correct to incorrectnucleotide incorporations when the nucleotides are present at equalconcentrations to compete for primer extension at the same site in thepolymerase-primer-template DNA binary complex. As proposed by Fersht,DNA polymerase fidelity can be calculated as the ratio of(k_(cat)/K_(m)) for the correct nucleotide and (k_(cat)/K_(m)) for theincorrect nucleotide; where k_(cat) and K_(m) are the familiarMichaelis-Menten parameters in steady state enzyme kinetics (Fersht, A.R. (1985) Enzyme Structure and Mechanism, 2nd ed., p 350, W. H. Freeman& Co., New York., incorporated herein by reference). Alternatively, inpre-steady state measurements, the ratio of (k_(pol)/K_(d)) for thecorrect and incorrect nucleotides can be used. In particularembodiments, a polymerase can have a fidelity value at least 100, 1000,10,000, 100,000, or 1 million, with or without a proofreading activity.

According to particular embodiments, a polymerase or other molecule canbe conformationally labeled. Conformational labeling of nucleic acidenzymes provides advantages for nucleic acid sequence analysis.Conformationally labeled molecules, and methods for making and usingthem, will be exemplified below with regard to labeled polymerases. Itwill be understood that other nucleic acid enzymes such as exonucleasesand reverse transcriptases can be made and used similarly.

Polymerases undergo conformational changes in the course of synthesizinga nucleic acid polymer. For example, polymerases undergo aconformational change from an open conformation to a closed conformationupon binding of a nucleotide. Thus, a polymerase that is bound to anucleic acid template and growing primer is in what is referred to inthe art as an “open” conformation. A polymerase that is bound to anucleic acid template, primer and a correctly base paired nucleotide isin what is referred to in the art as a “closed” conformation. At a moredetailed structural level, the transition from the open to closedconformation is characterized by relative movement within the polymeraseresulting in the “thumb” domain and “fingers” domain being closer toeach other. In the open conformation the thumb domain is further fromthe fingers domain, akin to the opening and closing of the palm of ahand. In various polymerases, the distance between the tip of the fingerand the thumb can change up to 10 angstroms between the “open” and“closed” conformations. The distance between the tip of the finger andthe rest of the protein domains can also change up to 10 angstroms. Itwill be understood that larger changes may also occur and can beexploited in a method set forth herein such that a change that isgreater than 10 angstroms can be detected. Furthermore, smaller changescan be detected including those that are less than about 10, 8, 6, 4, or2 angstroms so long as the change in distance is sufficient to bedetectable using the techniques employed.

In particular embodiments, a probe that is attached to a finger domaincan be attached to a residue at position 376 or residues within 5angstroms radius from position 376 of the Phi29 DNA polymerase and aprobe that is attached to the thumb or other domain can be attached to aresidue at position 535, 203, 510, 564, or residues within 5 angstromsradius from these positions of the Phi29 DNA polymerase. A molecularmodel showing some structural elements of Phi29 DNA polymerase and theirrelative locations is provided in FIG. 1 . For clarity, the polymerasestructure in the figure is reduced to elements that illustrate somerelevant features of finger domain movements. The conformation of thepolymerase in the open structure is shown in light grey. Upon theincoming nucleotide binding, the finger domain in the binary complex ofpolymerase and DNA moves closer to the thumb domain as indicated by thearrow labeled “close.” The resulting conformation for the finger domainis indicated by the dark grey helical structure. Some candidatepositions for attachment of probes are also labeled. It will beunderstood that homologous positions of these residues exemplified abovecan be used for other polymerases such as positions 550 and 744 in theKlenow Fragment.

In particular embodiments, a probe that is attached to a finger domaincan be attached to a residue at position 325 of Pol beta DNA polymerase.This position has been shown to be sensitive to the environmentalchanges caused by polymerase reactions. It will be understood thathomologous positions of the environmental sensitive residues exemplifiedabove can be used for other polymerases such as the position 514 of theT7 DNA polymerase and 845 of the Bacillus stearothermophilus DNApolymerase. Other useful polymerases and locations on polymerases forconformational labels are described in U.S. Pat. No. 6,908,763 and WO2010/068884 A2, each of which is incorporated herein by reference.

A change in conformation of a polymerase, for example, from an openconformation to a closed conformation, can be detected using aconformational probe. Any label or probe can be used that is responsiveto a change in the structure, shape or arrangement of amino acidresidues such as the changes that occur between the open and closedconformations of a polymerase. For example, an optical probe such as afluorescent probe can be used. The emission properties of a fluorescentprobe can change in response to changes in the local environment of thefluorophore.

A conformationally labeled enzyme, such as a polymerase or exonuclease,can include a pair of optical probes. For example, a fluorophore thatnormally has a detectable emission will be reduced or even preventedfrom emitting fluorescence when it comes into contact with a quencher.Accordingly, a conformationally labeled polymerase can have afluorophore that emits signal in either the open or closed conformationand that is quenched in the closed or open conformation, respectively.Similarly, a conformationally labeled polymerase can include a donor oracceptor fluorophore that forms one in a pair of fluorescence (orForster) resonance energy transfer (FRET) probes. One of the probes inthe FRET pair can be placed at the fingers domain and the other can beplaced at the thumb domain such that a change in conformation from theopen to closed conformation (or vice versa) can be detected based onchange in the amount of energy that is transferred to the acceptorand/or the amount of emission that is detected from the donor.

Exemplary fluorophores include, but are not limited to, fluorescentnanocrystals; quantum dots; d-Rhodamine acceptor dyes includingdichloro[R110], dichloro[R6G], dichloro[TAMRA], dichloro[ROX] or thelike; fluorescein donor dye including fluorescein, 6-FAM, or the like;Cyanine dyes such as Cy3B; Alexa dyes, SETA dyes, Atto dyes such as alto647N which forms a FRET pair with Cy3B and the like. Exemplary quenchersinclude, but are not limited to,DACYL(4-(4′-dimethylaminophenylazo)benzoic acid), Black Hole Quenchers(Biosearch Technologies, Novato, Calif.), Qxl quenchers (Anaspec,Freemont, Calif.), Iowa black quenchers, DABCYL, BHQ1, BHQ2, QSY7, QSY9,QSY21, QSY35, BHQO, BHQ1, BHQ2, QXL680, ATTO540Q, ATTO580Q, ATTO612Q,DYQ660, DYQ661 and IR Dye QC-1 quenchers. Fluorescent probes (includingdonors, acceptors, and quenchers) and methods for their use includingattachment to protein enzymes are described in Molecular Probes: TheHandbook (Invitrogen, Carlsbad Calif.), which is incorporated herein byreference. A fluorophore, quencher or other probe that is used in amethod or composition set forth herein can be an intrinsic probe that ispresent in a naturally occurring molecule being detected, such as atryptophan residue in a polymerase or exonuclease. Alternatively oradditionally, one can use a probe that is exogenous to a polymerase,exonuclease or other molecule being detected. Thus, in some embodimentssolely exogenous probes are detected such that endogenous probes are notdetected, in other embodiments solely endogenous probes are detectedsuch that exogenous probes are not detected and in some embodiments acombination of exogenous and endogenous probes are detected.

In particular embodiments, a split green fluorescent (GFP) protein canbe attached to a polymerase such that a portion of the GFP is fused tothe finger domain of the polymerase while the complementary portion ofthe GFP is fused to the thumb or other domains of the polymerase. Whenthe polymerase is in “open” conformation, the GFP fragments are farapart and fluorescence is inhibited or abolished. When the polymerase isin the “closed” conformation the GFP fragments are brought together andfluorescence appears or increases. The presence, absence, increase ordecrease of fluorescence can be detected in a method set forth herein.Other variants of GFP such as wavelength shifted variants can be usedsimilarly.

A probe can be attached to a polymerase, for example, via covalentlinkage. Alternatively or additionally, a probe can be attached toanother molecule that is in proximity to a polymerase, such that aconformational change in the polymerase causes a change in signal fromthe probe. For example, the polymerase can be attached to a solidsupport and the solid support can have a probe that is capable ofinteracting with the polymerase in a way that signals from the probechange in response to conformational changes of the polymerase. In aparticular embodiment, a probe can be attached site specifically to apolymerase by introducing cysteine residue at a desired location in thepolymerase and then modifying the polymerase with a probe having amoiety that reacts specifically with the sulfur group of cysteine, anexemplary reactive moiety being a reactive maleimide moiety. Anexemplary method for introducing a FRET probe pair (Cy3B and alto 647N)into a polymerase using site specific cysteine mutagenesis followed bychemical modification with dyes having maleimide moieties is describedin Santoso et al. Proc. Nat'l. Acad. Sci. USA 107:705-710 (2010), whichis incorporated herein by reference. Probes can also be introduced topolymerase, exonuclease or other nucleic acid enzyme by split inteins asdescribed in Yang et al. J. Am. Chem. Soc., 131:11644-11645 (2009),which is incorporated herein by reference. Probes can also be introducedto nucleic acid enzymes by genetically encoded unnatural amino acids.One example is described in Fleissner et al. Proc. Nat'l. Acad. Sci. USA106:21637-42 (2009), which is incorporated herein by reference.

Labels other than fluorescent labels can be used. For example, apolymerase or other nucleic acid enzyme can be labeled site specificallyby paramagnetic spin labels such as nitroxide, and the conformationalchanges of the enzyme can be detected by observing changes in therelaxation time of the spin label using electron paramagnetic resonanceand related techniques. Exemplary spin labels and techniques for theirdetection are described in Hubbell et al. Trends Biochem Sci. 27:288-95(2002), which is incorporated herein by reference.

A change in signal that is detected from an optical probe due to aconformational change can be, for example, a change in wavelength orintensity. In particular embodiments, the change in wavelength can be ashift in excitation wavelength maximum, change in excitation spectrum,shift in emission wavelength maximum, or change in emission spectrum.The intensity change can be an increase in extinction coefficient,decrease in extinction coefficient, increase in quantum yield, ordecrease in quantum yield. For example, a change in wavelength can bedetected due to a change in proximity or orientation between a donor andacceptor of a FRET pair. A change in intensity of signal can be detecteddue to a change in proximity between a fluorophore and quencher or achange in orientation between a fluorophore and quencher. A change inwavelength or intensity can also be detected due to a change instructure, protonation state, or environment of a fluorophore. Exemplarychanges in signal that can be detected in a method set forth hereininclude, without limitation, increased fluorescence resonance energytransfer (FRET, also referred to in the art as Förster resonance energytransfer), decreased FRET, increased fluorescence quenching or decreasedfluorescence quenching. Detection of other forms of energy transfer canalso be useful. Other changes in signal that can be detected from anoptical probe due to a conformational change include a change inemission polarization, decrease in excited state lifetime, or increasein excited state lifetime.

Other labels and methods for detecting conformational changes in apolymerase are described in U.S. Pat. No. 6,908,763 and WO 2010/068884A2, each of which is incorporated herein by reference. Although severalembodiments of the methods set forth herein can utilize conformationallylabeled polymerases, it will be understood that the label need notproduce a conformational signal change. Accordingly, the labels andlabeling techniques set forth herein can be used to label a polymeraseor other nucleic acid enzyme in a method where conformational signalchanges are not detected or distinguished.

In addition to the conformational changes set forth herein and otherwiseknown in the art, polymerases undergo several transitions in the courseof adding a nucleotide to a growing nucleic acid strand. The transitionscan be distinguished from each other, for example, by kineticcharacterization. As shown in FIG. 2 , distinguishable transitionsinclude, for example, the binding of primed nucleic acid to thepolymerase to form a polymerase-nucleic acid complex, the binding of anucleotide to the polymerase-nucleic acid complex to form an openpolymerase-nucleic acid-nucleotide ternary complex, the transition ofthe polymerase in the open polymerase-nucleic acid-nucleotide ternarycomplex to the closed polymerase′-nucleic acid-nucleotide ternarycomplex, catalytic bond formation between the nucleotide and nucleicacid in the closed polymerase′-nucleic acid-nucleotide ternary complexto form a closed polymerase′-extended nucleic acid-pyrophosphatecomplex, transition of the closed polymerase′-extended nucleicacid-pyrophosphate complex to an open polymerase-extended nucleicacid-pyrophosphate complex, release of pyrophosphate from the openpolymerase-extended nucleic acid-pyrophosphate complex to form an openpolymerase-extended nucleic acid complex, and eventual (i.e. optionallyafter several repetitions of nucleotide binding incorporation) releaseof the extended nucleic acid from the open polymerase-extended nucleicacid complex to form the uncomplexed polymerase. One or more of thetransitions that a polymerase undergoes when adding a nucleotide to anucleic acid can be detected using a conformationally labeledpolymerase. Similarly, the reverse transitions can be detected using aconformationally labeled polymerase, for example, to detect one or moretransitions that occur when a nucleotide is removed from a nucleic acidduring pyrophosphorolysis or hydrolysis. For example, time based orkinetic measurement of signals arising from a conformationally labeledpolymerase can be used to distinguish one transition from another.

In particular embodiments, time based or kinetic measurements of aconformationally labeled polymerase can be used to distinguish thespecies of nucleotide that is added to a nucleic acid. For example, atime based or kinetic measurement can be used to distinguish the speciesof nucleotide that is bound to a polymerase to form a polymerase-nucleicacid-nucleotide complex, to distinguish the species of nucleotide thatis involved in the transition of a polymerase in a polymerase-nucleicacid-nucleotide complex to a polymerase′-nucleic acid-nucleotidecomplex, to distinguish the species of nucleotide that is involved incatalytic bond formation between the nucleotide and a nucleic acid in apolymerase′-nucleic acid-nucleotide complex to form apolymerase′-extended nucleic acid-pyrophosphate complex, to distinguishthe species of nucleotide that is involved in transition of apolymerase′-extended nucleic acid-pyrophosphate complex to apolymerase-extended nucleic acid-pyrophosphate complex, or todistinguish the species of nucleotide that is involved in release ofpyrophosphate from a polymerase-extended nucleic acid-pyrophosphatecomplex to form a polymerase-extended nucleic acid complex.Alternatively or additionally, time based or kinetic measurements of aconformationally labeled polymerase can be used to distinguish thebinding and/or incorporation of a correctly Watson-Crick base-pairednucleotide from one that is incorrectly base-paired to the templatenucleic acid. Similarly, the binding and/or incorporation of amethylated nucleotide can be distinguished from one that is notmethylated, or the binding and/or incorporation of a ribonucleotide canbe distinguished from a deoxyribonucleotide.

A sequence of time based or kinetic measurements for a conformationallylabeled polymerase can be used to determine the sequence of a templatenucleic acid being used by the polymerase to synthesize a complementarystrand. It will be understood that the sequence of the template strandcan be inferred from the sequence of nucleotides incorporated into thestrand that is being extended. As such, determination of the sequence ofone strand will be understood to include determination of the sequenceof its complementary strand.

Similarly, time based or kinetic measurements of a conformationallylabeled polymerase can be used to distinguish the species of nucleotidethat is removed from a nucleic acid. For example, a time based orkinetic measurement can be used to distinguish the species of nucleotidethat is bound to a polymerase to form the intermediates set forth above(albeit in the reverse direction from that exemplified above fornucleotide addition) and shown in FIG. 2 . A sequence of time based orkinetic measurements for a conformationally labeled polymerase can beused to determine the sequence of a template nucleic acid that ishybridized to a strand being degraded by the polymerase.

The time duration of a single nucleotide's binding and incorporation bya polymerase can be at least 10 ms, 20 ms, 50 ms, 100 ms, 1000 ms, 5000ms, 10,000 ms, 60,000 ms or longer. Any of a variety of detectiontechniques known in the art can be used including, but not limited to,rapid kinetics analysis including stopped-flow and quench flowtechniques, CCD-based detection systems, EMCCD-based detection systems,ICCD-based detection systems, total internal reflectance fluorescence(TIRF)-based systems, or CMOS-based detection systems.

Detection can be carried out at ensemble or single molecule levels inreal time. Ensemble level detection includes detection that occurs in away that a population of molecules is detected such that individualmolecules in the population are not distinguished from each other. Thus,ensemble detection provides an average signal from the molecules in thepopulation. The population can be a colony or feature on a solid supportsuch as an array. The molecules in the population typically share commoncharacteristics, for example, a common sequence shared by severalnucleic acid molecules. In particular embodiments, ensemble detectionutilizes nucleotide analogs having reversible blocking groups as setforth in further detail below. At the ensemble level, the base callingcan be achieved by cycling one or two types of nucleotides each time.

Detection at a single molecule level includes detection that occurs in away that an individual molecule is distinguished. Thus, single moleculedetection provides a signal from an individual molecule that isdistinguished from one or more signals that may arise from a populationof molecules within which the individual molecule is present.

Any of a variety of nucleotide species can be useful in a method orcomposition set forth herein. For example, naturally occurringnucleotides can be used such as ATP, UTP, CTP, GTP, ADP, UDP, CDP, GDP,AMP, UMP, CMP, GMP, dATP, dTTP, dCTP, dGTP, dADP, dTDP, dCDP, dGDP,dAMP, dTMP, dCMP, and dGMP. Typically, dNTP nucleotides are incorporatedinto a DNA strand by DNA polymerases and NTP nucleotides areincorporated into an RNA strand by RNA polymerases. In particularembodiments, NTP nucleotides or analogs thereof can be incorporated intoDNA by a DNA polymerase, for example, in cases where the NTP, or analogthereof, is capable of being incorporated into the DNA by the DNApolymerase and where the rate or time duration for a DNA polymerasetransition using the NTP, or analog thereof, can be distinguished fromthe rate or time duration for the DNA polymerase transition usinganother nucleotide. Alternatively, dNTP nucleotides or analogs thereofcan be incorporated into RNA by an RNA polymerase, for example, in caseswhere the dNTP, or analog thereof, is capable of being incorporated intothe RNA by the RNA polymerase and where the rate or time duration for anRNA polymerase transition using the dNTP, or analog thereof, can bedistinguished from the rate or time duration for the RNA polymerasetransition using another nucleotide. Additionally, dNTP nucleotides oranalogs thereof can be incorporated into DNA from an RNA template by areverse transcriptase, for example, in cases where the dNTP, or analogthereof, is capable of being incorporated into the DNA from an RNAtemplate by a reverse transcriptase and where the rate or time durationfor a reverse transcriptase transition using the dNTP, or analogthereof, can be distinguished from the rate or time duration for thereverse transcriptase transition using another nucleotide. The relativedifference in rate or time duration can be a relative increase in therate, a relative increase in duration, a relative decrease in rate or arelative decrease in duration. The relative difference in intensity orother properties of probes, such as fluorescence correlation orpolarization, at the end point of the duration can also be used todistinguish different nucleotides. Additionally, the same principle canbe applied to distinguish methylated nucleotides in the template basedon the relative difference in rate or time duration or intensity orother properties at the end point of the duration for a DNA polymeraseincorporating a nucleotide opposite the methylated nucleotide in thetemplate.

Non-natural nucleotide analogs are also useful. Particularly usefulnon-natural nucleotide analogs include, but are not limited to, thosethat produce a detectably different rate or time duration for apolymerase transition that can be distinguished from the rate or timeduration for a polymerase transition with another nucleotide. Forexample, a non-natural nucleotide analog may usefully produce adetectably different rate or time duration for a polymerase transitionthat can be distinguished from the rate or time duration for the sametransition of the polymerase with another nucleotide such as a naturallyoccurring nucleotide. Exemplary nucleotide analogs that can be usedinclude, but are not limited to, dNTPaS; NTPaS; nucleotides havingunnatural nucleobases identified in Hwang et al., Nucl. Acids Res.34:2037-2045 (2006) (incorporated herein by reference) as ICS, 3MN, 7AI,BEN, DMS, TM, 2Br, 3Br, 4Br, 2CN, 3CN, 4CN, 2FB, 3FB, MM1, MM2 and MM3;or nucleotides having other non-natural nucleobases such as thosedescribed in Patro et al. Biochem. 48:180-189 (2009) (incorporatedherein by reference) which include 2-amino-1-deazapurine, 1-deazapurine,2-pyridine, hypoxanthine, purine, 6-Cl-purine, 2-amino-dA, 2-aminopurine or 6-C1-2-amino-purine or nucleotides having non-naturalnucleobases such as those described in Krueger et al. Chem Biol.16:242-8 (2009) (incorporated herein by reference) which include iso-G,iso-C, 5SICS, MMO2, Ds, Pa, FI, FB, dZ, DNB, thymine isosteres, 5-NI,dP, azole-carboxamide, xA, Im-No, Im-ON, J, A*, T*.

Non-natural nucleotide analogs having 5′ modifications are particularlyuseful. The non-natural nucleotide analog will typically have atriphosphate but can have more or fewer phosphates as set forthelsewhere herein. In particular embodiments, one or more of the alphaphosphate, beta phosphate or gamma phosphate of a non-natural nucleotideis covalently attached to a moiety other than oxygen. A moiety that isattached to a phosphate or otherwise present at the 5′ position canprovide a negative charge, a positive charge, metal-chelating activityor steric bulk. Exemplary moieties include, but are not limited to,amino acids, in the L-enantiomer form or R-enantiomer form, such ashistidine, aspartate, glutamate, tryptophan, phenylalanine, methionine,tyrosine, cysteine, glycine alanine, or proline; an amino group; achelated metal such as magnesium or manganese; a methyl group; a halogensuch as bromine, chlorine or iodine; a thiol group; an electronwithdrawing group; an electron donating group; an aromatic amine; or analiphatic amine. These and other moieties may be advantageous inembodiments where they provide an interaction with a polymerase, orother nucleic acid enzyme, that differs from the interaction that theenzyme has with a nucleotide lacking the moiety. As such, the presenceand absence of the moiety on respective nucleotide species can beexploited to distinguish the nucleotide species in a sequencing method,for example, based on the rate, time duration and/or intensity for aconformational signal change in a nucleic acid enzyme acting on thenucleotide species. See Example V below.

FIG. 3 provides further examples of non-natural nucleotide triphosphatesthat can be included in a composition set forth herein or used in amethod set forth herein. The examples shown in the figure contain adeoxyribose sugar moiety having a hydroxyl at the 3′ position. It willbe understood that the 3′ position can have a terminating group,reversible terminating group or other moiety such as those set forthelsewhere herein or otherwise known in the art. Furthermore other sugarmoieties can be used such as ribose or analogs known in the art. The“Base” moiety can be any of a variety of bases known in the artincluding, without limitation, adenine, thymine, uracil, cytosine,guanine or analogs thereof. As shown in the figure exemplarytriphosphate moieties include, but are not limited to,alpha-boranotriphosphate (FIG. 3A); alpha-phosphorothioate (FIG. 3B);beta,gamma-halomethylene bridged triphosphate (FIG. 3C) andgamma-phosphoamidate modified triphosphates (FIG. 3D). Exemplary Rgroups that can be present in the gamma-phosphoamidate modifiedtriphosphates are shown in FIG. 4 , wherein the “N-” moiety representsthe linkage to the gamma phosphate. As shown in FIG. 4 the R group canbe, for example, an electron withdrawing group, electron donating group,aromatic amine, aliphatic amine or other moiety. Exemplary gammaphosphoamidate-linked moieties and methods for their synthesis aredescribed in Mulder et al., Nucleic Acids Res. 33:4865-4873 (2005) andBerde et al., J. Biol. Chem. 254:12069-12073 (1979), each of which isincorporated herein by reference. Other examples of nucleotides having amoiety other than oxygen attached to a triphosphate moiety include thosehaving 1-alpha-thiol phosphate or 1-alpha-borano phosphate as furtherdescribed in Example III.

Another useful type of nucleotide is a caged nucleotide. An exemplarycaged nucleotide has a moiety with a photo-isomerizable double bond. Inparticular embodiments, a first isomer of the caged nucleotide has adifferent rate or time duration for a conformational signal change of apolymerase than a second isomer of the caged nucleotide. For example,the first isomer may readily bind to the polymerase and be incorporatedinto a nucleic acid under particular conditions whereas the secondisomer will not appreciably bind to the polymerase and/or beincorporated into the nucleic acid under the particular conditions.Azobenzene is a moiety that undergoes photo-isomerization whereby UVradiation causes trans to cis conversion and blue light causes cis totrans conversion. Other moieties that undergo photo-isomerization andconditions for their photo-isomerization are known in the art andinclude, for example, stilbene, and cinnamic acid.

A further example of a caged nucleotide is one having a moiety that isphoto-cleavable. In some embodiments, the presence of the moiety on thenucleotide alters (e.g. reduces or increases) the rate or time durationfor a conformational signal change of a polymerase compared to thenucleotide without the moiety. For example, a nucleotide lacking themoiety may readily bind to a polymerase and be incorporated into anucleic acid under particular conditions whereas the presence of themoiety will reduce or prevent binding to the polymerase and/orincorporation into the nucleic acid under the particular conditions.Exemplary photo-cleavable moieties include, but are not limited to(1-(4,5-dimethoxy-2-nitrophenyl)ethyl) ester (i.e. DMNPE) and(1-(2-nitrophenyl)ethyl) ester (i.e. NPE). See Meth. Enzymol.291:307-347 (1998), which is incorporated herein by reference.

A photo-isomerizable moiety or photo-cleavable moiety can be attached toa nucleotide at any of a variety of locations in the nucleotideincluding, but not limited to, the ribose moiety, a phosphate moiety, ora base moiety or other specific locations exemplified herein in thecontext of other nucleotide analogs. Furthermore, a photo-isomerizablemoiety or photo-cleavable moiety can be attached to one or morenucleotide species used in a method or reaction set forth herein. Forexample, such moieties can be present on a nucleotide analog having abase that pairs with adenine, thymine, guanine or cytosine. Mixtures ofnucleotides can be used that have different photo-isomerizable orphoto-cleavable moieties. The different moieties can be tuned forphotoreactions with different wavelengths of light. As such, individualnucleotide types can be activated (or deactivated) using differentwavelengths of light in order to provide light-gated control ofindividual nucleotide types in a reaction such as a sequencing reactionset forth herein.

Use of one or more caged nucleotide species can provide a means toinitiate, modulate or attenuate a reaction set forth herein. Forexample, one or more photo-isomerizable or photo-cleavable nucleotidespecies can be introduced to a reaction in an inactive conformation andsubsequently light activation can be used to initiate binding ofnucleotides to a polymerase or addition of the nucleotides to a nucleicacid by a polymerase. Thus, light activation can provide temporalcontrol of the start point for a reaction set forth herein.Alternatively or additionally, photo-isomerizable nucleotides that arein an active conformation can be inactivated by light to pause or stop apolymerization reaction. Stopping a reaction can be achieved byseparating reaction components from each other, for example by washingthe nucleotides away from a solid-phase attached nucleic acid. Such aseparation step need not be carried out and instead the reaction can beresumed by toggling the photo-isomerizable nucleotide to an active formto resume polymerization. As such, caged nucleotides provide a means toachieve light-gated control of a variety of reactions such as thesequencing methods set forth herein.

Light-gating is particularly useful for embodiments that use real-timedetection at a single molecule level. Single molecule reactions arestochastic by nature. Light-gating provides for temporal control ofdetection to coincide with initiation of the single molecule reactionthereby providing more accurate detection.

Although an advantage of light-gating is set forth above in regard toreal-time detection at a single molecule level, it will be understoodthat light gating is also useful for ensemble-level detection. Forexample, whether used for a single-molecule or ensemble levelembodiments, light gating can provide spatial control of a reaction.More specifically, a sample can contain a relatively large pool ofnucleotides and focused light can be delivered to a portion of a sampleto activate a sub-population of the nucleotides. Thus, repeatedactivation of a subpopulation of nucleotides can be used instead ofrepeated fluidic delivery steps.

Variants of polymerase can be engineered to incorporate and extendnatural or non-natural nucleotides at an appropriate or otherwisedesired speed to allow detection of differences in rate or time durationwhen different nucleotides are incorporated and extended.

A reaction composition or method can include one or more nucleotidespecies. For example, a reaction composition or method used for sequenceanalysis can include four different nucleotide species capable offorming Watson-Crick base pairs with four respective nucleotide speciesin a nucleic acid template being synthesized. Particular embodiments caninclude at least two different nucleotide species, at least threedifferent nucleotide species, at least four different nucleotidespecies, or more. At least two of the nucleotide species can benon-natural nucleotide analogs, at least three of the nucleotide speciescan be non-natural nucleotide analogs, or at least four of thenucleotide species can be non-natural nucleotide analogs. Thus areaction composition or method can include a mixture of naturalnucleotides and non-natural nucleotide analogs. Alternatively, areaction composition can lack natural nucleotides having instead onlynon-natural nucleotide analogs. The reaction can be carried out underconditions in which only non-natural nucleotide analogs are incorporatedinto a growing nucleic acid by a polymerase or other nucleic acidenzyme.

In some embodiments, a reaction composition or method can includenucleotide species that base-pair with no more than one nucleotidespecies in a nucleic acid template. For example, a method can be carriedout under conditions wherein different nucleotide species are contactedwith a polymerase and nucleic acid in separate, sequential reactions.Specifically, a nucleotide species that base-pairs with A can be addedin a first reaction, a nucleotide species that base-pairs with C can beadded in a second reaction, a nucleotide species that base-pairs with Tcan be added in a third reaction, and a nucleotide species thatbase-pairs with G can be added in a fourth reaction. The reactions arereferred to as first, second, third and fourth merely to illustrate thatthe reactions are separate but this does not necessarily limit the orderby which the species can added in a method set forth herein. Rather,nucleotide species that base-pair with A, C, T or G can be added in anyorder desired or appropriate for a particular embodiment of the methods.Typically in a sequencing method nucleotide species that base-pair withfour different nucleotide species in a given template nucleic acid areadded sequentially to complete a cycle of the sequencing method.However, it will be understood that fewer than four nucleotide additionscan be used in some embodiments. Furthermore, it will be understood thatmixtures of nucleotides that base-pair with more than one but no morethan 2, 3 or 4 nucleotide species can be used. Similarly, mixtures ofnucleotides that base-pair with more than two but no more than 3 or 4nucleotide species can be used. Or mixtures of nucleotides thatbase-pair with more than three but no more than 4 nucleotide species canbe used.

In particular embodiments, a method set forth herein can be carried outunder conditions wherein one or more of the nucleotides lack detectableprobes. A method can be carried out under conditions wherein all of thenucleotides lack detectable probes. For example, the nucleotide(s) canlack an exogenous probe. Exogenous probes include any probes that arenot present in the structure of a natural nucleotide including, forexample, an optical probe such as a fluorophore, optical quencher, orchromophore.

In particular embodiments, a method set forth herein can be carried outunder conditions wherein one or more of the nucleotides lack quenchers.A method can be carried out under conditions wherein all of thenucleotides lack quenchers. For example, the nucleotide(s) can lackquenchers that interact with a probe that is detected, such as a probeon a conformationally labeled enzyme or on a nucleic acid. Exemplaryquenchers include optical quenchers such as those that prevent or reducedetectable emission from a nearby fluorophore.

In particular embodiments, a method set forth herein can be carried outunder conditions wherein a nucleic acid, whether a template strand orits complement, lacks detectable probes. For example, a nucleic acid canlack an exogenous probe, such as those set forth above. Similarly, amethod set forth herein can be carried out under conditions wherein thenucleic acid lacks quenchers. For example, the nucleic acid can lackquenchers that interact with a probe that is detected such as a probe ona conformationally labeled enzyme or on a nucleotide.

In some embodiments, a method can be carried out under conditionswherein at least one nucleotide is undetectable including, for example,a condition wherein all of the nucleotides are undetectable.Alternatively or additionally, a method can be carried out underconditions wherein a nucleic acid, whether a template strand or itscomplement, is undetectable. A nucleotide or nucleic acid can beundetectable due to the use of a detection device or detection mode thatis incapable of detecting signals produced by the nucleotides or nucleicacids. For example, an optical device can include an optical filter thatrejects optical signals in a range produced by the nucleotides and/ornucleic acids. Alternatively or additionally, an optical device can beconfigured such that it does not substantially excite nucleotides and/ornucleic acids in a way that optically detectable signals are produced.

A method set forth herein can be carried out in solution or on a solidsupport. A solution-phase method will be understood to be one where allcomponents that participate in a reaction are in solution, thecomponents including, for example, a nucleic acid, nucleic acid enzymeand nucleotide. A solid-phase reaction is one where one or more of thecomponents occur in or on a solid support. For example, a nucleic acid,nucleic acid enzyme or nucleotide can be in or on a solid support duringthe course of a solid-phase reaction. A nucleic acid that is attached tothe solid support can be a template nucleic acid such as one that iscopied by a polymerase, a primer nucleic acid such as one that isextended by a polymerase, or a double stranded nucleic acid such as onethat is acted upon by a polymerase, exonuclease or other nucleic acidenzyme.

Any of a variety of solid-support materials can be used in a method orcomposition set forth herein. Useful materials include, for example,those that are separable from each other such as beads, particles,microspheres, or chromatographic supports; and those that form acontinuous material such as a flow cell, microchip or other chip,microscope slide or other planar surface, or the like. Particularlyuseful supports are those used for microarrays. Useful materials for amicroarray or other solid support include, but are not limited to,glass; modified glass; functionalized glass; plastics such as acrylics,polystyrene and copolymers of styrene and other materials,polypropylene, polyethylene, polybutylene, polyurethanes, Teflon, or thelike; polysaccharides; nylon; nitrocellulose; resins; silica;silica-based materials such as silicon or modified silicon; carbon;metal; inorganic glass; optical fiber bundles, or any of a variety ofother polymers. Useful substrates include those that allow opticaldetection, for example, by being translucent to energy of a desireddetection wavelength and/or do not produce appreciable backgroundfluorescence at a particular detection wavelength.

A reaction component can be attached to a solid support by methods knownin the art. In some embodiments, a component such as a nucleic acid canbe synthesized on a solid support by sequential addition of nucleotideunits directly on the solid support. Methods known in the art forsynthesis of a variety of nucleic acids on solid supports can be used.Alternatively, components can be synthesized or otherwise obtainedfirst, and then covalently attached to a solid support. The componentscan be attached to functional groups on a solid support. Functionalizedsolid supports can be produced by methods known in the art and, ifdesired, obtained from any of several commercial suppliers for beads andother supports having surface chemistries that facilitate the attachmentof a desired functionality by a user. Exemplary surface chemistries thatare useful in the invention include, but are not limited to, aminogroups such as aliphatic and aromatic amines, carboxylic acids,aldehydes, amides, chloromethyl groups, hydrazide, hydroxyl groups,sulfonates or sulfates. If desired, a component can be attached to asolid support via a chemical linker. Such a linker can havecharacteristics that provide, for example, stable attachment, reversibleattachment, sufficient flexibility to allow desired interaction withanother reaction component, or to avoid undesirable binding reactions.By way of example, a surface with an array of attached oligo dTmolecules can be used to immobilize sheared genomic DNA pieces that havepoly A tail transferred by Terminal transferase. Further exemplarymethods that can be used in the invention to attach polymer probes to asolid support are described in Pease et al., Proc. Natl. Acad. Sci. USA91(11):5022-5026 (1994); Khrapko et al., Mol Biol (Mosk) (USSR)25:718-730 (1991); Stimpson et al., Proc. Natl. Acad. Sci. USA92:6379-6383 (1995) or Guo et al., Nucleic Acids Res. 22:5456-5465(1994), each of which is incorporated herein by reference.

A reaction component can be attached to a support in a way that providesdetection at a single molecule level or at an ensemble level. Forexample, a population of nucleic acids can be attached to a solidsupport in a way that conformationally labeled polymerases that interactwith individual nucleic acid molecules in the population can bedistinguished from conformationally labeled polymerases that interactwith other nucleic acid molecules on the support. Single moleculedetection can also be achieved with a population of conformationallylabeled polymerases that is attached to a solid support in a way thatsignals arising from a particular polymerase can be distinguished fromsignals arising from other polymerases on the support. Reactioncomponents can be separated from each other on a solid support due tosurface features or contours such as those that form wells, posts,channels or the like. Alternatively or additionally, separation can beachieved by providing spacing between molecules that is greater than theresolution of a particular detection device that is in use.

Ensemble detection can be achieved for reaction components that areattached to a surface to form colonies or clusters for ensembledetection. For example, one or more colonies each containing severalconformationally labeled polymerases can be attached to a surface.Colonies of nucleic acids can be attached to a surface using methodsknown in the art such as bridge amplification or emulsion PCR. Usefulbridge amplification methods are described, for example, in U.S. Pat.No. 5,641,658; U.S. Patent Publ. No. 2002/0055100; U.S. Pat. No.7,115,400; U.S. Patent Publ. No. 2004/0096853; U.S. Patent Publ. No.2004/0002090; U.S. Patent Publ. No. 2007/0128624; and U.S. Patent Publ.No. 2008/0009420. Another useful method for amplifying nucleic acids ona surface is rolling circle amplification (RCA), for example, asdescribed in Lizardi et al., Nat. Genet. 19:225-232 (1998) and US2007/0099208 A1, each of which is incorporated herein by reference.Exemplary emulsion PCR methods are described in Dressman et al., Proc.Natl. Acad. Sci. USA 100:8817-8822 (2003), WO 05/010145, or U.S. PatentPubl. Nos. 2005/0130173 or 2005/0064460, each of which is incorporatedherein by reference in its entirety.

Whether occurring in solution phase or solid phase formats, a polymeraseextension method can be carried out by a single delivery of nucleotidesor by multiple deliveries of nucleotides. In an exemplary embodiment,the former configuration can include a single delivery of severaldifferent species of nucleotides such that the polymerase is able to addseveral nucleotides to a growing nucleic acid strand. In such a methodmultiple nucleotide deliveries are not necessary to achieve extension ofa primer by at least 2, 3, 5, 10, 50, 100, 250, 500, 1000, 10000 or morenucleotides. Typically, four different nucleotide species will bedelivered, but if desired, fewer than four can be delivered. Deliveringnucleotides one at a time is one way for base calling at the ensemblelevel.

In a particular embodiment, one or more blocked nucleotide species canbe delivered such that single base extension occurs. In an exemplaryembodiment of the multiple nucleotide addition format, reversiblyblocked nucleotides can be delivered each time. Deblocking and washingsteps can be carried out between nucleotide addition steps. Typically achemically reactive deblocking group is used; however a photo-sensitiveblock can be used for fast deblocking by light. Exemplary modificationsthat can be used to render a nucleotide reversibly blocked and stepsthat can be used for cyclical addition of blocked nucleotides bypolymerase extension are described in U.S. Pat. Nos. 7,427,673;7,414,116; and 7,057,026 and PCT publications WO 91/06678 and WO07/123744, each of which is incorporated herein by reference.

Reversibly blocked nucleotides can be particularly useful for detectionat an ensemble level. The blocking group on the nucleotide can providequantization or synchronization of extension events occurring for apopulation of conformationally labeled polymerases. An example isillustrative as follows. A solid support is provided having a cluster ofidentical nucleic acids having a common template sequence.Conformationally labeled polymerases are bound to templates to formpolymerase-template species in the cluster. Reversibly blockednucleotides of a particular type (e.g. A, C, T or G) are delivered tothe cluster of polymerase-template species thereby resulting in a singleextension for each species in the cluster. The extension event at eachcluster is detected as an average signal from the conformationallylabeled polymerases in the cluster. Following the detection event, thereversible blocking groups are removed from the nucleotides that wereincorporated into the cluster and then the nucleotide delivery anddetection events are repeated. In this way, the extension eventsoccurring for several template-polymerase species in the cluster aresynchronized or quantized with respect to each nucleotide additionevent. As such the sequence of signals from the cluster can be detectedin order to determine the template sequence for the nucleic acids of thecluster. Reversibly blocked nucleotides are also useful for embodimentsthat employ detection at single molecule resolution. As such, the methodsteps exemplified above are not intended to be limited to ensemble-baseddetection.

Also provided herein is a method of determining a sequence ofnucleotides for a nucleic acid template. The method can include thesteps of contacting the nucleic acid template with a conformationallylabeled exonuclease under conditions wherein the conformationallylabeled exonuclease catalyzes sequential removal of nucleotide speciesfrom the nucleic acid template, wherein the sequential removal of eachdifferent nucleotide species produces a conformational signal changefrom the conformationally labeled exonuclease and wherein the rate ortime duration for the conformational signal change is distinguishablefor each different nucleotide species that is removed; detecting aseries of changes in the signal from the conformationally labeledexonuclease under the conditions; and determining the rates or timedurations for the changes in the signal for the series of changes in thesignal from the conformationally labeled exonuclease, therebydetermining the sequence of nucleotides for the nucleic acid template.The conformationally labeled exonuclease can be a polymerase havingexonucleolytic or pyrophosphorolysis activity.

In particular embodiments, a nucleic acid that is sequenced using aconformationally labeled exonuclease can contain one or more species ofmodified nucleotide subunits. Individual species of nucleotide subunitscan contain a unique moiety that interacts with an exonuclease duringremoval from the nucleic acid to produce a rate or time duration for aconformational signal change that is distinguishable from the rate ortime duration produced by the other types of nucleotide species that areremoved from the nucleic acid. The nucleic acid can contain at least 1,at least 2, at least 3 or at least 4 modified nucleotide species.Exemplary species include those having modified alpha-phosphate moietiessuch as those set forth above, shown in FIGS. 3 and 4 or otherwise knownin the art.

This disclosure further provides a method of determining a sequence ofnucleotides for a nucleic acid template. The method can include thesteps of contacting the nucleic acid template with a conformationallylabeled polymerase and at least four different nucleotide species underconditions wherein the conformationally labeled polymerase catalyzessequential addition of the nucleotide species to form a nucleic acidcomplement of the nucleic acid template, wherein the sequential additionof each different nucleotide species produces a conformational signalchange from the conformationally labeled polymerase and wherein the rateor time duration for the conformational signal change is distinguishablefor each different nucleotide species; detecting a series of changes inthe signal from the conformationally labeled polymerase under theconditions; determining the rates or time durations for the changes inthe signal, thereby determining the sequence of nucleotides for thenucleic acid template; contacting the nucleic acid template with aconformationally labeled exonuclease under conditions wherein theconformationally labeled exonuclease catalyzes sequential removal ofnucleotide species from the nucleic acid template, wherein thesequential removal of each different nucleotide species produces aconformational signal change from the conformationally labeledexonuclease and wherein the rate or time duration for the conformationalsignal change is distinguishable for each different nucleotide speciesthat is removed; detecting a series of changes in the signal from theconformationally labeled exonuclease under the conditions; anddetermining the rates or time durations for the changes in the signalfor the series of changes in the signal from the conformationallylabeled exonuclease.

The conformationally labeled polymerase and the conformationally labeledexonuclease can be the same molecular species or different molecularspecies. The species can differ from each other, for example, in theirprimary amino acid sequences, the location of one or more labels, thechemical structure of one or more labels, the presence or absence of aprotein domain (such as an exonuclease domain), or the presence orabsence of a modification that substantially influences exonuclease orpolymerase activity.

A method of sequencing a nucleic acid can include a polymerase phase forsequencing-by-synthesis of the nucleic acid followed by an exonucleasephase for sequencing-by-degradation of the nucleic acid. The degradationphase can advantageously provide a proofreading function for thesynthesis phase. In proofreading embodiments, the exonuclease phase canprovide a resolution that is equivalent to or lower than the resolutionof the polymerase phase. For example, a sequencing-by-synthesis phasethat uses a conformationally labeled polymerase can be used to obtain asingle nucleic acid sequence that resolves the positions of all fournucleotide species and a subsequent sequencing-by-degradation phase canbe used to determine the locations of at most 1, 2 or 3 of the fournucleotide species. Proofreading can be achieved by aligning thelocations of the nucleotide species identified in thesequencing-by-degradation phase with their locations as identified inthe sequencing-by-synthesis phase, whereby any misalignment wouldindicate a potential error in the sequence obtained in thesequencing-by-synthesis phase.

For proofreading embodiments, a sequencing-by-synthesis phase thatincorporates a nucleotide species having a modified alpha-phosphatemoiety is particularly useful because the modified alpha-phosphatemoiety if present in the synthesized nucleic acid can provide a rate ortime duration for the conformational signal change that isdistinguishable from that produced by other nucleotide units in thenucleic acid. Accordingly, it can be advantageous to include at leastone modified alpha-phosphate moiety in a method of sequencing. It can befurther advantageous to include no more than 2 or 3 nucleotides havingthe modified alpha-phosphate moiety, for example, to allow thenucleotide having the modified alpha-phosphate moiety to bedistinguished from other nucleotides in a proofreading embodiment.

In a further embodiment a method of determining a sequence ofnucleotides for a nucleic acid sample is provided. The method caninclude the steps of providing an array of nucleic acid templates,wherein the nucleic acid templates include nucleotide sequence fragmentsof the nucleic acid sample; contacting the array of nucleic acidtemplates with conformationally labeled polymerases and at least fourdifferent nucleotide species under conditions wherein theconformationally labeled polymerases catalyze sequential addition of thenucleotide species to form nucleic acid complements of the nucleic acidtemplates, wherein the sequential addition of each different nucleotidespecies produces a conformational signal change from theconformationally labeled polymerase and wherein the rate or timeduration for the conformational signal change is distinguishable foreach different nucleotide species; detecting a series of changes in thesignal from the conformationally labeled polymerase under the conditionsand at individual locations of the array; and determining the rates ortime durations for the changes in the signal at the individual locationsof the array, thereby determining the sequence of nucleotides for thenucleic acid sample.

Multiplex detection can be achieved using a microarray format. Examplesof array formats that can be used in the invention include, withoutlimitation, those described in Butte, Nature Reviews Drug Discov.1:951-60 (2002) or U.S. Pat. Nos. 5,429,807; 5,436,327; 5,561,071;5,583,211; 5,658,734; 5,837,858; 5,874,219; 5,919,523; 6,136,269;6,287,768; 6,287,776; 6,288,220; 6,297,006; 6,291,193; 6,346,413;6,416,949; 6,482,591; 6,514,751 and 6,610,482; and WO 93/17126; WO95/11995; WO 95/35505; EP 742 287; and EP 799 897, each of which isincorporated herein by reference. Other useful array formats are thosein which separate substrates are located in solution or on a surfaceincluding, without limitation, those having beads as described, forexample, in U.S. Pat. Nos. 6,023,540, 6,200,737, 6,327,410 and6,355,431; US Pat. Pub. No. 2002/0102578; and PCT publications WO98/40726, WO 98/50782, WO 99/18434 and WO 00/63437, each of which isincorporated herein by reference. For embodiments including bead-basedarrays, the arrays can be made, for example, by adding a solution orslurry of the beads to a substrate containing attachment sites for thebeads. Beads can be loaded into the wells of a substrate, for example,by applying energy such as pressure, agitation or vibration, to thebeads in the presence of the wells. Methods for loading beads onto arraysubstrates that can be used in the invention are described, for example,in U.S. Pat. No. 6,355,431, which is incorporated herein by reference.

A useful method for making arrays is photolithography-based polymersynthesis. For example, Affymetrix™ GeneChip™ arrays can be synthesizedin accordance with techniques sometimes referred to as VLSIPS™ (VeryLarge Scale Immobilized Polymer Synthesis) technologies. Some aspects ofVLSIPS™ and other microarray manufacturing methods and techniques havebeen described in U.S. Pat. Nos. 5,143,854, 5,242,974, 5,252,743,5,324,633, 5,445,934, 5,744,305, 5,384,261, 5,405,783, 5,424,186,5,451,683, 5,482,867, 5,491,074, 5,527,681, 5,550,215, 5,571,639,5,578,832, 5,593,839, 5,599,695, 5,624,711, 5,631,734, 5,795,716,5,831,070, 5,837,832, 5,856,101, 5,858,659, 5,936,324, 5,968,740,5,974,164, 5,981,185, 5,981,956, 6,025,601, 6,033,860, 6,040,193,6,090,555, 6,136,269, 6,269,846, 6,022,963, 6,083,697, 6,291,183,6,309,831 and 6,428,752, each of which is incorporated herein byreference.

A spotted array can also be used. An exemplary spotted array is aCodeLink′ Array available from Amersham Biosciences. CodeLink′ ActivatedSlides are coated with a long-chain, hydrophilic polymer containingamine-reactive groups. This polymer is covalently cross-linked to itselfand to the surface of the slide. Attachment of reaction components canbe accomplished through covalent interaction between the amine-modifiednucleic acid or protein and the amine reactive groups present in thepolymer. Components can be attached at discrete locations using spottingpens. Another array that is useful in the invention can also bemanufactured using inkjet printing methods such as SurePrint™ Technologyavailable from Agilent Technologies. Such methods can be used tosynthesize nucleic acids in situ or to attach pre-synthesized reactioncomponents having moieties that are reactive with a substrate surface.

The size of an array can vary depending on the desired use of the array.Arrays useful in the invention can have complexity that ranges fromabout 2 different reaction sites to many millions, billions or higher.The density of an array can be from 2 to as many as a billion or moredifferent reaction sites per square cm. Very high density arrays areuseful in the invention including, for example, those having at leastabout 10,000,000 reaction sites/cm², including, for example, at leastabout 100,000,000 reaction sites/cm², 1,000,000,000 reaction sites/cm²,up to about 2,000,000,000 reaction sites/cm² or higher. High densityarrays can also be used including, for example, those in the range fromabout 100,000 reaction sites/cm² to about 10,000,000 reaction sites/cm².Moderate density arrays useful in the invention can range from about10,000 reaction sites/cm² to about 100,000 reaction sites/cm² Lowdensity arrays are generally less than about 10,000 reaction sites/cm².

A method of determining a sequence of nucleotides for a nucleic acid(i.e. a nucleic acid sequencing method) can be carried out using avariety of protocols. Typically, a nucleic acid sequencing method iscyclic due to repetitious addition of nucleotide or oligonucleotideunits to a growing nucleic acid polymer or repetitious removal ofnucleotide or oligonucleotide units from a nucleic acid polymer. Anucleic acid sequencing protocol can also, but need not, includerepeated cycles of manipulation, each cycle of manipulations includingone or more steps. For example, each cycle of manipulations can includeone or more steps that result in detection of a single nucleotide thatis added to a growing nucleic acid. Sequencing methods that utilize aconformationally labeled polymerase and reversible terminators generallyprovide for detection of a single nucleotide per cycle. Similarly, eachcycle of manipulations can include one or more steps that result indetection of a single nucleotide that is removed from a shrinking (orfragmenting) nucleic acid. Alternatively, some protocols providedetection of multiple nucleotides per cycle, for example, by exploitingthe cyclic nature of nucleic acid enzymes and detection of theiractivity. In some embodiments, this detection of multiple nucleotides iscarried out using real-time detection of conformational signal changesfollowing a single fluidic manipulation. Several exemplary sequencingprotocols are set forth below for illustration.

A first exemplary nucleic acid sequencing protocol can be carried outfor a sequencing reaction that includes, inter alia, a primed nucleicacid template, conformationally labeled polymerase and a set of fourdifferent nucleotide species. The four different nucleotide speciesdiffer in their base composition and in the rate or time duration for aconformational signal change that occurs for the polymerase when eachspecies is incorporated into the primed nucleic acid template. The fourdifferent nucleotide species are extendable, for example, lackingterminator groups at the 3′ position of the ribose moiety. The protocolcan include delivery of all four nucleotides to the sequencing reactionsuch that the four nucleotides are present simultaneously and real timedetection of the conformational signal changes occurring in thepolymerase, whereby the identity of each nucleotide species isdistinguished according to the rate or time duration for the respectiveconformational signal change. A single nucleic acid sequence thatresolves the positions of all four nucleotide species can be obtainedusing the first exemplary protocol.

A second exemplary nucleic acid sequencing protocol can be carried outfor a sequencing reaction that includes, inter alia, a primed nucleicacid template, conformationally labeled polymerase and a set of fourdifferent nucleotide species. The four different nucleotide speciesdiffer in their base composition. A first nucleotide of the set differsfrom the other three nucleotides in the rate or time duration for aconformational signal change that occurs for the polymerase when thefirst nucleotide is incorporated into the primed nucleic acid template.The other three nucleotides are not necessarily distinguished from eachother based on the rate or time duration for a conformational signalchange that occurs for the polymerase when they are incorporated intothe primed nucleic acid template. The four different nucleotide speciesare extendable, for example, lacking terminator groups at the 3′position of the ribose moiety. The protocol can include delivery of allfour nucleotides to the sequencing reaction such that the fournucleotides are present simultaneously and real time detection of theconformational signal changes occurring in the polymerase, whereby theidentity of the first nucleotide species is distinguished according tothe rate or time duration for the respective conformational signalchange and whereby incorporation of the other three nucleotides isdetected based on the conformational signal change. A single nucleicacid sequence that resolves the positions of a single nucleotide speciescan be obtained using the second exemplary protocol.

A third exemplary nucleic acid sequencing protocol can be carried out asset forth above for the second exemplary nucleic acid sequencingprotocol with the exception that a second set of nucleotides is used inplace of the first set of nucleotides that was used in the secondexemplary protocol. The second set of nucleotides can differ from thefirst set of nucleotides in the identity of the nucleotide that differsfrom the other three nucleotides in the rate or time duration for theconformational signal change. For example, the second set of nucleotidescan include dATP, dTTP, dCTP and γANSdGTP whereas the first set ofnucleotides included dATP, dTTP, γANSdCTP and dGTP. Again a singlenucleic acid sequence that resolves the positions of a single nucleotidespecies (i.e. cytosine) can be obtained using the third exemplaryprotocol. Comparison of the results of the second and third sequencingprotocol can provide a low resolution sequence of the nucleic acidtemplate in which the position of two nucleotides (i.e. guanine andcytosine) is resolved.

A low resolution sequence can provide a useful scaffold for sequencealignment as set forth in U.S. Patent Publ. Nos. 2010/0173303 and2010/0279882, each of which is incorporated herein by reference.Alternatively, the third exemplary nucleic acid sequencing protocol canbe repeated using a third and fourth set of nucleotides whereby twodifferent nucleotides are respectively distinguishable. Comparing theresults of all four sequencing protocols can provide a single nucleicacid sequence that resolves the positions of all four nucleotide speciesin the nucleic acid template. In some embodiments only three of theabove protocols need be run such that the locations of 3 types of basesare detected. In this scenario, the location of the fourth type of basecan be inferred from the data derived for the other 3 types of bases.

A fourth exemplary nucleic acid sequencing protocol can be carried outfor a sequencing reaction that includes, inter alia, a primed nucleicacid template, conformationally labeled polymerase and a set of fourdifferent nucleotide species, wherein each nucleotide species has areversible terminator moiety. The four different nucleotide speciesdiffer in their base composition and in the rate or time duration for aconformational signal change that occurs for the polymerase when eachspecies is incorporated into the primed nucleic acid template. Theprotocol can include a nucleotide delivery step whereby all fournucleotides are present in the sequencing reaction simultaneously, adetection step whereby the identity of each nucleotide species isdistinguished according to the rate or time duration for a respectiveconformational signal change in the polymerase, a deblocking step toremove the reversible terminator moieties, and repetition of theaforementioned steps. Wash steps can be carried out to remove reactioncomponents between one or more of the steps. Generally a singlenucleotide will be identified for each repetition. After severalrepetitions, a single nucleic acid sequence that resolves the positionsof all four nucleotide species can be obtained using the fourthexemplary protocol.

A fifth exemplary nucleic acid sequencing protocol can be carried outfor a sequencing reaction that includes, inter alia, a primed nucleicacid template, conformationally labeled polymerase and a singlenucleotide species, wherein the nucleotide species has a reversibleterminator moiety. The protocol can include separate nucleotide deliverysteps whereby each of four nucleotides are present in the sequencingreaction individually, a detection step whereby incorporation of thenucleotide species is determined according to the rate or time durationfor a conformational signal change in the polymerase, a deblocking stepto remove the reversible terminator moieties, and repetition of theaforementioned steps with the other three nucleotides. Wash steps can becarried out to remove reaction components between one or more of thesteps. The four different nucleotide species differ in their basecomposition but not necessarily in the rate or time duration for aconformational signal change that occurs for the polymerase when eachspecies is incorporated into the primed nucleic acid template. The fourphase nucleotide delivery and detection protocol can be repeated forseveral cycles. Generally, a single nucleotide will be identified foreach cycle of four phases. After several repetitions, a single nucleicacid sequence that resolves the positions of all four nucleotide speciescan be obtained using the fifth exemplary protocol.

A sixth exemplary nucleic acid sequencing protocol can be carried outfor a sequencing reaction that includes, inter alia, a primed nucleicacid template, conformationally labeled polymerase and a set of fourdifferent nucleotide species, wherein each nucleotide species has areversible terminator moiety. A first nucleotide of the set differs fromthe other three nucleotides in the rate or time duration for aconformational signal change that occurs for the polymerase when it isincorporated into the primed nucleic acid template. The other threenucleotides are not necessarily distinguished from each other based onthe rate or time duration for a conformational signal change that occursfor the polymerase when they are incorporated into the primed nucleicacid template. The protocol can include a nucleotide delivery stepwhereby all four nucleotides are present in the sequencing reactionsimultaneously; a detection step, whereby the identity of the firstnucleotide species is distinguished from the other three nucleotidespecies according to the rate or time duration for a respectiveconformational signal change in the polymerase and whereby incorporationof the other three nucleotides is detected based on the conformationalsignal change; a deblocking step to remove the reversible terminatormoieties; and repetition of the aforementioned steps. Wash steps can becarried out to remove reaction components between one or more of thesteps. After several repetitions, a single nucleic acid sequence thatresolves the positions of a single nucleotide species can be obtainedusing the second exemplary protocol.

A seventh exemplary nucleic acid sequencing protocol can be carried outas set forth above for the sixth exemplary nucleic acid sequencingprotocol with the exception that a second set of nucleotides is used inplace of the first set of nucleotides that was used in the sixthexemplary protocol. The second set of nucleotides can differ from thefirst set of nucleotides in the identity of the nucleotide that differsfrom the other three nucleotides in the rate or time duration for theconformational signal change. For example, the second set of nucleotidescan include rtATP, rtdTTP, rtdCTP and γANSrtGTP (“rt” refers to thepresence of a reversible terminator) whereas the first set ofnucleotides included rtATP, rtTTP, γANSrtCTP and rtGTP. Again a singlenucleic acid sequence that resolves the positions of a single nucleotidespecies (i.e. cytosine) can be obtained using the seventh exemplaryprotocol. Comparison of the results of the sixth and seventh sequencingprotocol can provide a low resolution sequence of the nucleic acidtemplate in which the position of two nucleotides (i.e. guanine andcytosine) is resolved.

An eighth exemplary nucleic acid sequencing protocol can be carried outfor a sequencing reaction that includes, inter alia, a primed nucleicacid template, conformationally labeled polymerase and a singlenucleotide species, wherein the nucleotide species is selected fromnaturally occurring nucleotides including dATP, dCTP, dGTP, dTTP, anddUTP. The protocol can include separate nucleotide delivery stepswhereby each of four nucleotides are present in the sequencing reactionindividually, a detection step whereby incorporation of the nucleotidespecies is determined according to the rate or time duration for aconformational signal change in the polymerase. Wash steps can becarried out to remove reaction components between one or more of thesteps. The four phase nucleotide delivery and detection protocol can berepeated for several cycles. Generally, a single nucleotide will beidentified for each cycle of four phases. After several repetitions, asingle nucleic acid sequence that resolves the positions of all fournucleotide species can be obtained using the fifth exemplary protocol.When this exemplary protocol is carried out in a single moleculedetection mode, the numbers of the homopolymers in the nucleic acid canbe determined by counting the number of times the conformational changehas happened as demonstrated by probes.

As set forth previously, a low resolution sequence can provide a usefulscaffold for sequence alignment as set forth in U.S. Patent Publ. Nos.2010/0173303 and 2010/0279882, each of which is incorporated herein byreference. Alternatively, the seventh exemplary nucleic acid sequencingprotocol can be repeated using a third and fourth set of nucleotideswhereby two different nucleotides are respectively distinguishable.Comparing the results of all four sequencing protocols can provide asingle nucleic acid sequence that resolves the positions of all fournucleotide species in the nucleic acid template.

A ninth exemplary nucleic acid sequencing protocol can be carried outfor a sequencing reaction that includes, inter alia, a primed nucleicacid template, conformationally labeled polymerase and a first set oftwo different nucleotide species. The two different nucleotide speciesin the first set differ in their base composition and in the rate ortime duration for a conformational signal change that occurs for thepolymerase when each species is incorporated into the primed nucleicacid template (e.g. γANSdGTP and dATP). The two different nucleotidespecies in the first set are extendable, for example, lacking terminatorgroups at the 3′ position of the ribose moiety. The protocol can includedelivery of the first set to the sequencing reaction such that the twonucleotides are present simultaneously and the conformational signalchanges occurring in the polymerase are detected in real time, wherebythe identity of each nucleotide species is distinguished according tothe rate or time duration for the respective conformational signalchange. An optional wash step can be carried out to remove unreactednucleotides from the primed nucleic acid template. Then a second set oftwo different nucleotide species can be contacted with the primednucleic acid template and a conformationally labeled polymerase, whereinthe two nucleotides of the second set are present simultaneously and theconformational signal changes occurring in the polymerase are detectedin real time and the identity of each nucleotide species in the secondset is distinguished according to the rate or time duration for therespective conformational signal change. The second set of nucleotidespecies includes two different nucleotide species that differ from eachother in their base composition and also differ in the rate or timeduration for a conformational signal change that occurs for thepolymerase when each species is incorporated into the primed nucleicacid template (e.g. γANSdCTP and dTTP). The nucleotides in the secondset differ in base composition from the two nucleotides that were in thefirst set of nucleotide species; however, the nucleotides in the firstset need not be distinguishable from the nucleotides of the second setwith respect to the rate or time duration for a conformational signalchange that occurs for the polymerase since the two set of nucleotidesare delivered and detected in separate steps. Although the pairs ofnucleotide species are delivered in separate steps, a single nucleicacid sequence that resolves the positions of all four nucleotide speciescan be obtained using the ninth exemplary protocol.

As exemplified by the protocols above, a polymerase extension method canbe carried out by a single delivery of nucleotides or by multipledeliveries of nucleotides. In an exemplary embodiment, the formerconfiguration can include a single delivery of several different speciesof nucleotides such that the polymerase is able to add severalnucleotides to a growing nucleic acid strand. In such a method multiplenucleotide deliveries are not necessary to achieve extension of a primerby at least 2, 3, 5, 10, 50, 100, 250, 500, 1000, 10000 or morenucleotides. However, if desired multiple nucleotide deliveries can beperformed and each delivery can include several different species ofnucleotides such that the polymerase is able to add several nucleotidesto a growing nucleic acid strand. Typically, four different nucleotidespecies will be delivered, but if desired, fewer than four can bedelivered.

In a particular embodiment, one or more blocked nucleotide species canbe added such that single base extension occurs. One or more blockednucleotide species can be used in an embodiment whereby a polymeraseextension method is carried out by a single delivery of nucleotides orby multiple deliveries of nucleotides. In an exemplary embodiment of themultiple nucleotide delivery format, several different species ofreversibly blocked nucleotides can be present simultaneously in areaction cycle. Deblocking and washing steps can be carried out betweennucleotide addition steps. Thus, the sequencing procedure can be carriedout as a series of repeated cycles of nucleotide delivery, detection anddeblocking. The nucleotides can be delivered simultaneously orsequentially during each cycle. Washes can be carried out between stepsof each cycle as desired to remove unwanted reactants or products frombeing present in subsequent cycles or subsequent steps of a currentcycle.

Typically, nucleotides having different bases can be distinguished in amethod set forth herein according to different moieties present on therespective nucleotide species. For example a dCTP can have a gammaphosphoamidate-linked moiety that is not present on a dGTP, therebyallowing incorporation of the dCTP into a nucleic acid to bedistinguished from incorporation of the dGTP using a conformationallylabeled polymerase. However if desired, a first and second population ofnucleotides having a common base can have different moieties such thatthe two populations can be distinguished from each other in a method setforth herein. This is demonstrated by Example V, where incorporation ofgamma-ANS-dTTP by a conformationally labeled polymerase is to bedistinguished from incorporation of dTTP by the same polymerase.

Similarly, detection of time domain differences between differentnucleotides when they are being incorporated or cleaved by a polymerasecan be used for the detection of any of a variety of modifications ofnucleic acids. For example, a methylated nucleotide can have a timedomain signature that is unique from all other unmethylated nucleotides.Methods similar those set forth above and in the Examples below can beused to distinguish methylated from unmethylated nucleotides in anucleic acid molecule.

Particular embodiments provide a method of determining nucleotidesequences which can optionally include the steps of (a) providing anarray of different nucleic acid templates; (b) providing a mixture ofnucleotide species, the mixture including (i) at least four differentnucleotide species, (ii) at least one of the four different nucleotidespecies having a reversible terminator moiety, and (iii) at least two ofthe four different nucleotide species having an extendible 3′ hydroxylmoiety; (c) contacting the array of nucleic acid templates withconformationally labeled polymerases and the mixture of nucleotidespecies under conditions wherein the conformationally labeledpolymerases catalyze sequential addition of the nucleotide species toform nucleic acid complements of the nucleic acid templates, wherein thesequential addition of each different nucleotide species produces aconformational signal change from the conformationally labeledpolymerase, wherein the rate or time duration for the conformationalsignal change is distinguishable for the at least two nucleotide specieshaving the extendible 3′ hydroxyl moiety, and wherein a plurality of thenucleic acid complements incorporate the at least one nucleotide speciesthat has the reversible terminator moiety; (d) removing the reversibleterminator moiety; (e) detecting a series of changes in the signal fromthe conformationally labeled polymerase at individual locations of thearray; and (f) determining the sequence of nucleotides for the nucleicacid sample from the series of changes in the signal from theconformationally labeled polymerase.

As exemplified for the above embodiment, at least one nucleotide specieshaving a reversible terminator moiety can be used in combination withnucleotide species that have an extendible 3′ hydroxyl moiety. Forexample, different nucleotide species, each having a different basemoiety capable of complementing one of four respective base species in atemplate nucleic acid, can be used. In this example at least one of thefour different nucleotide species can have a reversible terminatormoiety while the three other species have an extendible 3′ hydroxylgroup. Incorporation of the three extendible species into a nucleic acidcan be distinguished as set forth herein, for example, based ondifferences in rate or time duration for signal changes that occur in aconformationally labeled polymerase that incorporates the nucleotidesinto the nucleic acid. The incorporation of the nucleotide specieshaving the reversible terminator can be distinguished from the otherthree nucleotide species based on the termination in extension thatoccurs for the nucleic acid. For embodiments that use a conformationallylabeled polymerase, the termination can be detected as a pause in thedetection of conformational changes in the polymerase. The terminationcan be reversed using a deblocking reagent as set forth previouslyherein and, if desired, extension can then be resumed. Thus, thetermination that resulted from incorporation of the nucleotide specieshaving the reversible terminator can be distinguished from the otherthree nucleotide species based on the resumption of extension aftertreatment of the nucleic acid with the deblocking reagent.

For ease of description the example above describes use of a singlenucleotide species having a reversible terminator moiety in combinationwith 3 other nucleotide species having an extendible 3′ hydroxyl moiety.It will be understood that more than one reversibly blocked nucleotidespecies can be used with extendible nucleotide species in a method setforth herein. For example, different nucleotide species, each having adifferent base moiety capable of complementing one of four respectivebase species in a template nucleic acid, can include at least one, two,three, or four species having a reversible terminator moiety. Inparticular embodiments, a mixture of four different nucleotide speciescan include no more than 1 species having a reversible terminator moietyand at least 3 different species having an extendible 3′ hydroxyl; nomore than 2 different species having a reversible terminator moiety andat least 2 different species having an extendible 3′ hydroxyl; or nomore than 3 different species having a reversible terminator moiety andat least 1 species having an extendible 3′ hydroxyl. Of course, someembodiments can include 4 different species having a reversibleterminator moiety. The combinations of nucleotide species exemplifiedabove can be provided in a mixture that is contacted with a nucleic acidsample or the nucleotides can be provided to a nucleic acid sampleindividually, for example, in a stepwise manner.

Methods that employ a nucleotide species having a reversible terminatormoiety are typically carried out in repeated cycles. In embodiments thatutilize a combination of nucleotides having reversible terminatormoieties and extendible 3′ hydroxyl groups, it is possible to extend atleast some nucleic acid sequences with several nucleotide additions.Extension will continue due to the incorporation of extendiblenucleotides up until a reversibly terminated nucleotide is incorporated.The incorporation of each nucleotide species can be detected, and inmany embodiments the species of nucleotide that is incorporated can bedistinguished, as set forth elsewhere herein. A deblocking step can beused to remove reversible blocking moieties from an extended nucleicacid. For example, a deblocking reagent can be used to remove achemically labile blocking moiety or light can be used to remove aphotolabile blocking moiety. Extension and detection steps can then becarried out again. Several repetitions of the extension, detection anddeblocking steps can be carried out for a nucleic acid, for example, todetermine the sequence of nucleotides for the nucleic acid.

In particular embodiments, at least one species of nucleotide that isused in a method set forth herein can be present at a concentration thatis substantially lower than the concentration of another nucleotidespecies used in the method. As a result, the nucleotide species that ispresent at low concentration can produce a signal change in aconformationally labeled polymerase that is distinguishable from thechange occurring for other nucleotide species that are present at higherconcentration. The distinction can be made, for example, based onaltered rate or time duration for a signal change. The concentration ofone or more nucleotide species used in a method can be selected toresult in a low branching ratio. Branching is the binding of theappropriate nucleotide to a polymerase without productive incorporationof the nucleotide into a nucleic acid molecule by the polymerase.

Particular embodiments of methods of determining nucleotide sequencescan use a mixture of nucleotide species, wherein the mixture includes(i) at least four different nucleotide species, (ii) at least one of thefour different nucleotide species having a reversible terminator moiety,(iii) at least two of the four different nucleotide species having anextendible 3′ hydroxyl moiety, and (iv) at least one of the fourdifferent nucleotide species being present at a substantially lowerconcentration than the concentration of any other nucleotide species inthe mixture. Specifically, the nucleotide that is present at thesubstantially lower concentration can be present at a concentration thatproduces a distinguishable rate or time duration for the conformationalsignal change.

The following examples are intended to illustrate but not limit thepresent invention.

Example I Polymerase Engineering

A panel of polymerases from family A and family B including Klenowfragment, T7 polymerase, Bst polymerase, 9° N polymerases, KOD, RB69polymerase, Phi29 polymerase and/or Bsu polymerase, is surveyed. NativeCys residues are replaced by Ser, Val, or Ala using known site-directedmutagenesis techniques. Molecular modeling based on existing crystalstructures such as those described in Berman et al. EMBO J. 26:3494-3505 (2007), and Kamtekar et al. EMBO J. 25: 1335-1343 (2006), eachof which is incorporated herein by reference, are used to identify pairsof locations where the relative movements caused by the conformationalchanges of the polymerases between the open and the closed conformationsare maximum and easily detectable by probes. Candidates are shown inFIG. 2 . The amino acid residues at the chosen pairs of locations arechanged to Cys residues by site-directed mutagenesis.

The resulting double Cys mutants are then expressed in E coli andpurified using affinity chromatography via a genetically fused His-tag,GST-tag, and/or Heparin affinity column.

Labeling of the double Cys mutants with sulfhydryl specific probes, suchas fluorophores, follows manufacturers' recommendations and/ortechniques described in Santoso et al. Proc. Nat'l. Acad. Sci. USA107:705-710 (2010), incorporated herein by reference. In short, thedouble-Cys mutant proteins are labeled by sequential addition of twomaleimide fluorophores. The two fluorophores compose a FRET pair. Thefirst maleimide fluorophore is added to protein at 1:1 molar ratio andincubated at 22° C. at 2 hours. Labeling occurs predominantly at the Cysresidue that is more surface exposed and has higher reactivity. Thesecond maleimide fluorophore is then added at high molar excess for anadditional 10 hours. The reaction is stopped by addition ofdithiothreitol to 1 mM, and the unincorporated fluorophores are removedby gel filtration on a Bio-Spin 30 column.

The activity of the doubly-labeled polymerases is assessed by measuringthe rate of nucleotide addition to a DNA primer terminus by chemicalquench methods as described in Joyce et al. Biochemistry 47:6103-6116(2008), and Johnson K A. Methods Enzymol 249:38-61 (1995), each of whichis incorporated herein by reference.

Optionally, the active doubly labeled polymerase is then used as thebackbone for further mutagenesis, to generate polymerase variants thatcan incorporate and extend unnatural nucleotides at a desirable rate.

Molecular modeling based on crystal structures is exploited to identifythe locations of the polymerases where mutations can be made to allowfaster or slower incorporation and extension of unnatural nucleotides.Residue(s) identified as targets for replacement are replaced with aresidue or residues selected using energy minimization modeling,homology modeling, and/or conservative amino acid substitutions todetermine best case selections derived from known best substitutiontables. Such strategies are well known in the art as described, forexample, in Bordo, et al. J Mol Biol 217: 721-729 (1991), which isincorporated herein by reference. These strategies can be used togenerate a library of mutants with desired substitutions, which can thenbe assayed for incorporation and extension rates relative to a parentalpolymerase, as described below. Generation of libraries is welldescribed in the art such as Hayes, et al. Proc Natl Acad Sci. USA 99:15926-15931 (2002), which is incorporated herein by reference.

Example II Creation of Nucleotide Sets for Sequencing

Stopped-flow fluorescence kinetic analysis is used in accordance withtechniques known in the art such as those described in Johnson, et al.The Enzymes XX, 1-61 (1992), which is incorporated herein by reference.Measurements are performed on polymerases such as those engineered asset forth above in Example I to obtain the rates of both incorporationand extension of natural and unnatural nucleotides.

A panel of unnatural nucleotides, including for example those set forthherein above, is tested for one or more polymerases until fourdistinctive rates are obtained for the four nucleotides corresponding todATP, dCTP, dGTP, and dTTP. The selected polymerase mutant and fourunnatural nucleotides are used for single molecule detection andsequencing methods.

Example III Detection of Nucleotide Incorporation Into a Nucleic AcidUsing FRET-Labeled Polymerase

The Klenow Fragments (KF) was produced as follows. KF was mutated toreplace cysteine 907 with glycine, to replace leucine 744 (in thefingers domain) with cysteine and to replace lysine 550 (in the thumbdomain) with cysteine, thereby producing K550C/L744C/C907G KF. Standardmutagenesis techniques were used to produce K550C/L744C/C907G KF asdescribed in the manual of the QuikChange® site-directed mutagenesis kitfrom Stratagene/Agilent (La Jolla, Calif.). The K550C/L744C/C907G KF wascloned into the pET15b plasmid using the NdeI and BamHI restrictionsites, and expressed in E. coli BL21(DE3) cells. The K550C/L744C/C907CKF was purified as described in Joyce, et al. Biochemistry, 47 (23):6103-6116 (2008), which is incorporated herein by reference. Thepurified K550C/L744C/C907C KF was chemically modified using thethiol-reactive maleimide to introduce the Alexa488 fluorescent donor dyeat cysteine 550

and to introduce the Alexa532 fluorescence acceptor dye at cysteine 744,

thereby producing Alexa 488 and 532 dual-labeled KF. The thiol-reactivedye labeling protocol is described in Molecular Probes: The Handbook(Invitrogen, Carlsbad Calif.), which is incorporated herein byreference.

Conformational changes in the dual labeled KF enzyme from the open toclosed state can be detected based on fluorescence resonance energytransfer (FRET) between the AF488 fluorescent donor dye on the thumbdomain and AF532 fluorescence acceptor dye on the fingers domain. The KFenzyme can be bound to a primer-template nucleic acid complex in an openconformation. In the open conformation the AF488 fluorescent donor dyewhen excited with light at a wavelength of 495 nm will emit fluorescenceat a wavelength of 519 nm. However, upon binding of an appropriatenucleotide to the dual labeled KF-template-primer complex the duallabeled KF enzyme changes from the open state to the closed state. Theclosed state brings the AF488 fluorescent donor dye on the thumb domaininto proximity with the AF532 fluorescence acceptor dye on the fingerdomain such that the AF488 fluorescent donor dye when excited transfersenergy to the AF532 fluorescence acceptor dye. This FRET results in adetectable emission from the AF532 fluorescence acceptor dye at awavelength of 531 nm. As such, binding of the appropriate nucleotide canbe detected as increased emission at 554 nm wavelength. Furthermore,differential time durations of the closed state can be used todistinguish different nucleotides that are incorporated into the primerby dual labeled KF.

The real-time fluorescence change from dual labeled KF was measured inan Applied Photophysics SX20 stopped-flow spectrometer by monitoringchanges in AF 488 dye fluorescence following the mixing of thePolI(KF)-DNA binary complex with a nucleotide in the reaction buffercontaining 10 mM Tris-HCl, pH 8.0, 50 mM NaCl, 1 mM dithiothreitol and10 mM MgCl₂.

As shown in FIG. 5 the incorporation of natural nucleotides into theprimer could be detected based on FRET signals from dual labeled KF.However, the time durations of the FRET signal for the naturalnucleotides dATP, dGTP, dTTP and dCTP were very similar.

In contrast, FRET duration could be modulated by using non-naturalnucleotides. As shown in FIG. 6 , the FRET durations measured for theincorporation of dCTP, 1-alpha-thiol-dCTP and 1-alpha-borano-dCTP weresubstantially different. Similarly, the FRET durations measured for theincorporation of dTTP differed from those measured for incorporation ofdUTP (see FIG. 7 ).

As shown in FIG. 8 , the incorporation of a correct dGTP nucleotide at aposition that was complementary to cytosine could be distinguished fromthe incorrect incorporation of dCTP at the same position based ondifferences in FRET duration.

This example demonstrates that the incorporation of a nucleotide into agrowing primer nucleic acid can be detected using a conformationallylabeled polymerase having a pair of FRET probes. This example furtherdemonstrates that the incorporation of different nucleotides by aconformationally labeled polymerase can be distinguished based ondifferences in the time duration for a conformational signal changeproduced by the conformationally labeled polymerase. This example alsodemonstrates that the time duration for a conformational signal changeproduced by the conformationally labeled polymerase can be modulated byusing non-natural nucleotide analogs.

Example IV Detection of Nucleotide Incorporation Into a Nucleic Acid bya Polymerase Having an Environment-Sensitive Dye

5-TAMRA Pol β was prepared as follows. Human Pol β was mutated toreplace Tryptophan 325 (in the fingers domain) with cysteine to produceW325C Pol β. Standard mutagenesis techniques were used to produce W325CPol β as described in Example III. The W325C Pol β was cloned in thepET15b plasmid, expressed in E. coli BL21(DE3) cells and purified asdescribed in Dunlap and Tsai, Biochemistry, 41 (37): 11226-11235 (2002),which is incorporated herein by reference. The purified W325C Pol β waschemically modified to introduce the 5-TAMRA fluorescent dye at cysteine325, thereby producing 5-TAMRA Pol β.

Conformational changes in the 5-TAMRA Pol β enzyme from the open toclosed state can be detected based on a fluorescent emission change fromthe environmentally sensitive 5-TAMRA dye. The spectral and emissionintensity changes from the 5-TAMRA dye when the polymerase changes tothe closed state. The 5-TAMRA Pol β enzyme can be bound to aprimer-template nucleic acid complex in an open conformation. In theopen conformation the 5-TAMRA dye when excited with light at awavelength of 542 nm will emit fluorescence at a wavelength of 568 nm.However upon binding of an appropriate nucleotide to the 5-TAMRA Polβ-primer-template binary complex, the 5-TAMRA Pol β enzyme changes fromthe open state to the closed state causing the 5-TAMRA dye to experiencea different environment. This change in environment results in adetectable fluorescent emission change from the TAMRA dye at thewavelength of 568 nm. As such, binding of the appropriate nucleotide canbe detected as a fluorescent emission change at 568 nm wavelength.Furthermore, differential time durations of the closed state can be usedto distinguish different nucleotides that are incorporated into theprimer by 5-TAMRA Pol R.

The real-time fluorescence change from 5-TAMRA Pol β was measured in theApplied Photophysics SX20 stopped-flow spectrometer by monitoringchanges in 5-TAMRA dye fluorescence following the mixing of the 5-TAMRAPol β-DNA binary complex with a nucleotide in a reaction buffercontaining 50 mM Tris-HCl, pH 7.5, 50 mM KCl, 1 mM Dithiothreitol and 5mM MgCl₂ as described in Example III.

As shown in FIG. 9A, incorporation of a correct dCTP nucleotide into the5-TAMRA Pol β could be detected based on the wavelength shift for the5-TAMRA dye. Furthermore, as shown in FIG. 9B, incubation of thepolymerase with incorrect nucleotides did not yield a substantial shiftin 5-TAMRA emission.

Different nucleotides, dATP, dTTP, dCTP and dGTP produced differentamplitudes and durations for the 5-TAMRA emission shift whenincorporated at an appropriate position in a template-bound primer (seeFIG. 10 ). Furthermore, non-native nucleotide analogs 1-alpha-thiol-dCTPand 1-alpha-borano-dCTP could be distinguished from each other as well(see FIG. 11 ). As such the different nucleotide species could bedistinguished using 5-TAMRA Pol β as a reporter.

This example demonstrate that the incorporation of a nucleotide into agrowing primer nucleic acid can be detected using a conformationallylabeled polymerase having an environmentally sensitive dye. This examplefurther demonstrates that the incorporation of different nucleotides bythe conformationally labeled polymerase can be distinguished based ondifferences in the time duration for a conformational signal changeproduced by the conformationally labeled polymerase. This example alsodemonstrates that the time duration for a conformational signal changeproduced by the conformationally labeled polymerase can be modulated byusing non-natural nucleotide analogs.

Example V Detection of Gamma-Phosphate Modified Nucleotide IncorporationInto a Nucleic Acid Using Conformationally-Labeled Polymerase

Dual labeled KF was produced as described in Example III. FRET from duallabeled KF was measured by stopped flow kinetics also as described inExample III. 5-TAMRA Pol β was prepared as described in Example IV. Thereal time fluorescence change from 5-TAMRA Pol β was measured by stoppedflow kinetics also as described in Example IV.

Gamma-ANS-dTTP was synthesized as described in Mulder et al., NucleicAcids Res. 33:4865-4873 (2005) and Berde et al., J. Biol. Chem.254:12069-12073 (1979), each of which is incorporated herein byreference.

As shown in FIG. 12 the AF488 fluorescence duration measured for theincorporation of dTTP and Gamma-ANS-dTTP by dual labeled KF weresubstantially different. Similarly, incorporation of dTTP andGamma-ANS-dTTP by 5-TAMRA Pol β could be distinguished from each other(see FIG. 13 ).

This example demonstrates that the incorporation of a gamma-phosphatemodified nucleotide by a conformationally labeled polymerase can bedistinguished from incorporation of a natural nucleotide based ondifferences in the time duration for a conformational signal changeproduced by the conformationally labeled polymerase.

Example VI Light-Gated Sequencing

FIG. 14A shows a diagrammatic representation of a light-gated sequencingreaction. A sample is provided having conformationally labeled DNApolymerase, primer-template DNA complexes, a first nucleotide species(caged) and a second nucleotide species.

Individual primer-template DNA complexes are separated from each otheron an array surface to allow conformational signal changes forpolymerases bound to each complex to be detected at a single-moleculelevel.

Caged nucleotide species 1 has a photo-cleavable NPE moiety attached tothe 5′ gamma phosphate. The NPE moiety prevents caged nucleotide species1 from binding to the conformationally labeled polymerase. The NPEmoiety is photo-cleavable by irradiation with UV light to produceuncaged nucleotide species 1. Uncaged nucleotide species 1 is able tobind the conformationally labeled polymerase and can be incorporatedinto the primer strand of the primer-template complex. Nucleotidespecies 2 has a gamma-ANS moiety. The time duration for theconformational signal change of the conformationally labeled polymeraseis longer for nucleotide species 2 than for uncaged nucleotide species1.

A low power light pulse is delivered to the sample such that asubpopulation of caged nucleotide species 1 becomes uncaged (byphoto-cleavage of the NPE moiety). The light pulse initiates thepolymerase extension reaction because the base moiety of the uncagednucleotide species complements the first position of the templatestrand. The time duration for conformational signal changes at eacharray feature (i.e. an individual polymerase bound to an individualprimer-template complex) is detected. A sequence of time durations,measured for an individual array feature, is shown in FIG. 14B. As shownin the figure, incorporation of the two species can be distinguishedbased on different time durations for the signal changes they cause inthe polymerase. Specifically, the sequence of incorporations followingthe first uncaging light pulse is 1111122211 (where “1” representsuncaged nucleotide species 1 and “2” represents nucleotide species 2).

As shown in FIG. 14A, the extension reaction will terminate when thesubpopulation of uncaged nucleotide species 1 has been substantiallydepleted and the polymerase arrives at a location in the template thatcomplements species 1. The extension reaction can be resumed bydelivering a second low power light pulse to the sample such that asecond population of caged nucleotide species 1 become uncaged.

For clarity of description the example of FIG. 14B shows the extensionreaction going until depletion of uncaged nucleotide followed bydelivery of the second light pulse. It is however, desirable in someembodiments to deliver light pulses prior to depletion of reagents. Forexample, light pulses can be delivered on a preset schedule. The rate ofdelivery for light pulses can also be controlled in response to afeedback loop that determines the extension rate for one or morepolymerases in a sample. A rate of extension that is below a thresholdrate would be indicative of reduced nucleotide concentration, in whichcase the duration or intensity of light pulses can be increased to bringthe extension rate above the threshold. Conversely, a rate of extensionthat is above a ceiling rate would be indicative of excess nucleotideconcentration, in which case the duration or intensity of light pulsescan be decreased to bring the extension rate below the ceiling.

For purposes of illustration this example has been described withrespect to a system having two nucleotide species, one of which iscaged. Similar systems can use 3, 4 or more nucleotides and of thosenucleotides 1, 2, 3, 4 or more of the species can be caged. Furthermore,the different nucleotide species can be uncaged using differentwavelengths of light.

Example VII Design, Creation, Production and Analysis of aConformationally Labeled Polymerase

Family B RB69 DNA polymerase was modified to create the RB69V410CE766Cconjugate. The structure of RB69 has been solved at 2.8 Å resolution inthe open and closed states as described in Wang et al., Cell89:1087-1099 (1997), which is incorporated herein by reference. Here thestructures were analyzed revealing that the 0-helix in the finger domainundergoes a 60° rotation upon nucleotide binding, which corresponds to achange of about 27 Å in the α-α carbon distance between the amino acidsat positions 410 and 766. In the open position the amino acids are 69.4Å apart and in the closed position they move to within 41.5 Å of eachother. Förster Resonance Energy Transfer (FRET) is a method which can beused to detect a polymerase conformational change. The Förster radius(Ro) is the radius at which energy transfer between a donor dye and anacceptor dye is equal to 0.5. Energy transfer scales as approximately1/r⁶ (where r=distance between a donor and acceptor dye) which makes itadvantageous to position dyes at the Ro. The Ro for most standardcommercial dyes is in the 50-70 Å range.

Cysteine residues are generally reactive toward a wide range ofcommercially available dyes. Therefore, RB69 was mutated as follows.Native Cysteine (C) residues were mutated to other natural amino acidsat the following positions C41A, C57V, C456V, C609L, C671A, C748A,C801T, C845V. Also, native residues at positions 510 and 766 weresubstituted with C (V510C and E766C, respectively) to provide locationson the polymerase for site specific labeling. The resulting polymeraseis referred to as RB69V510CE766C.

RB69V510CE766C was expressed in BL21(DE3) cells. Cells were grown inTerrific Broth (TB) at 37° until an OD of 0.8. The temperature was thenreduced to 18 degrees and protein expression was induced with 0.5 mMIPTG. Cells were grown for an additional 16 hours. Following harvesting,the resulting cell pellet was lysed using a microfluidizer and thelysate treated with PEI to precipitate cellular DNA/RNA. PEI treatmentwas followed by ammonium sulfate (AS) fractionation. The (AS) pelletcontaining RB69V510CE766C was resolubilized and loaded onto a heparincolumn. RB69 was eluted from the heparin column using a salt gradient.The resulting peak fractions were pooled, concentrated, and loaded ontoa Superdex 200 column for size exclusion chromatography. The resultingpeak fractions were pooled, and purity confirmed by SDS-page gel.

Sulfhydryl reactive dyes were covalently attached to the cysteineresidues of purified RB69V510CE766C as follows. Purified RB69V510CE766Cwas first treated with 10 mM dithiothreitol (DTT) for 30 min at roomtemperature to reduce disulfide bonds. Next, RB69V510CE766C was purifiedby fractionating with a Sephadex-25 column using the following buffer:50 mM ACES pH 7.0, 1 M NaCl, 1 mM EDTA, and 0.01% w/v Tween-20. Thefractions with RB69V510CE766C were identified by measuring the 280 nmabsorption for each fraction. The RB69V510CE766C concentration wasestimated using the 6280=150000 M⁻¹cm⁻¹ (Wang et al., Biochemistry43:3853-3861 (2004), incorporated herein by reference). Then,conjugation reactions were carried out to label RB69V510CE766C with oneof the following donor/acceptor pairs: Cy3/AF647 or CF555/AF647(Cy3-maleimide was obtained from GE Healthcare, CF555-maleimide wasobtained from Biotium and AF647-maleimide was obtained from LifeTechnologies). The dyes were in 100×molar excess relative to theRB69V510CE766C concentration and various donor/acceptor ratios weretitrated to achieve nearly a 1:1:1 RB69V510CE766C:donor:acceptor ratio.The dye conjugation reaction proceeded for 12 h at 4° C. TheRB69V510CE766C polymerase conjugate was purified using two steps. First,a Sephadex-25 column was used to separate the labeled conjugate fromfree dyes using 50 mM Tris pH 7.5, 1 M NaCl, 1 mM EDTA, 0.01% w/vTween-20. The fractions containing the labeled conjugates wereidentified by measuring the absorption spectrum for each fraction. Thefractions with product were pooled and dialyzed against 50 mM Tris pH7.5, 1 M NaCl, 1 mM EDTA, 0.01% w/v Tween-20 for >4 h at 4° C. Theabsorption spectrum for final product was used to determine the degreeof dyes per polymerase using the dye supplier's specifications.

FIG. 15 shows an absorption spectrum for purified RB69V510CE766CCy3AF647conjugate. The ratio of RB69V510CE766C:Cy3:AF647 was determined to be1:1:0.7 from the spectrum.

Functionality of the Cy3AF647 and CF555AF647 conjugates ofRB69V510CE766C were confirmed using a FRET-based primer extension assayas follows. Extension was carried out on a nucleic acid duplex composedof an extendable 3′-OH primer and a template with a 7 base overhang. Theextendable sequence was TGGAACG. In addition, the template contained anAF488 dye at the 5′ position. The extension reaction mix was composed of50 mM ACES pH 7.2, 50 mM NaCl, 10 mM DTT, 10 mM Mg²⁺, 1 μM dTTP, 1 μMdGTP, 1 μM dATP and 0.1 μM Cy3-dCTP. Upon extension, the Cy3 quenchesthe AF488. The control reactions were carried out under the sameconditions except that dTTP was removed to prevent extension. ASpectraMax M5 plate reader was used to monitor the AF488 quenching overtime. FIG. 16 shows a plot of AF488 fluorescence vs. time for theextension reaction. The plot confirms that both conjugates werefunctional in extending a primed nucleic acid.

Throughout this application various publications, patents and patentapplications have been referenced. The disclosures of these publicationsin their entireties are hereby incorporated by reference in thisapplication in order to more fully describe the state of the art towhich this invention pertains.

The term “comprising” is intended herein to be open-ended, including notonly the recited elements, but further encompassing any additionalelements.

Although the invention has been described with reference to the examplesprovided above, it should be understood that various modifications canbe made without departing from the invention. Accordingly, the inventionis limited only by the claims.

What is claimed is:
 1. A method of determining nucleotide sequences,comprising: providing an array of nucleic acid templates; contacting thearray of nucleic acid templates with a polymerase that isconformationally labeled with an environment-sensitive optical probe,and at least four different nucleotide species under conditions wherethe conformationally labeled polymerase catalyzes sequential addition ofthe nucleotide species to form a nucleic acid complement of the nucleicacid template, wherein the sequential addition of each differentnucleotide species produces a conformational signal change from theconformationally labeled polymerase, wherein the amplitude and/or rateor time duration for the conformational signal change is distinguishablebased upon which of the nucleotide species is being added; detecting aseries of changes in the signal from the conformationally labeledpolymerase at individual locations of the array; and determining thesequence of nucleotides for the nucleic acid complement from the seriesof changes in the signal from the conformationally labeled polymerase.2. The method of claim 1, wherein the array of nucleic acid templatesare attached to a solid support.
 3. The method of claim 2, wherein thesolid support is a flow cell, a microarray, a microchip, or beads. 4.The method of claim 1, wherein the environment-sensitive optical probeis an environment-sensitive fluorescent dye.
 5. The method of claim 1,wherein conformational changes of the polymerase from an open to closedstate change the fluorescent emission of the environment-sensitivefluorescent dye.
 6. The method of claim 1, wherein the amplitude and/orrate or time duration for the conformational signal change isdistinguishable based upon whether a correct nucleotide or incorrectnucleotide is added.
 7. The method of claim 1, wherein the amplitudeand/or rate or time duration for the conformational signal change isdistinguishable based upon which nucleotide is added.
 8. The method ofclaim 7, wherein the nucleotide is a naturally occurring nucleotide. 9.The method of claim 7, wherein the nucleotide is a non-naturalnucleotide analog.
 10. The method of claim 1, wherein the at least fourdifferent nucleotide species comprise four different naturally occurringnucleotides.
 11. The method of claim 1, wherein the at least fourdifferent nucleotide species comprise at least one non-naturalnucleotide analog.
 12. The method of claim 1, wherein the rate or timeduration for the conformational signal change is measured using stoppedflow kinetics.
 13. The method of claim 1, wherein the conformationalsignal change comprises a change in wavelength or intensity.
 14. Themethod of claim 1, further comprising contacting the array of nucleicacid templates comprising the nucleic acid complements with aconformationally labeled exonuclease under conditions wherein theconformationally labeled exonuclease catalyzes sequential removal ofnucleotide species from the nucleic acid complements, wherein thesequential removal of each different nucleotide species produces aconformational signal change from the conformationally labeledexonuclease and wherein the rate or time duration for the conformationalsignal change is distinguishable for at least one different nucleotidespecies that is removed; detecting a series of changes in the signalfrom the conformationally labeled exonuclease under the conditions andat the individual locations of the array; and determining the rates ortime durations for the changes in the signal for the series of changesin the signal from the conformationally labeled exonuclease.
 15. Themethod of claim 14, wherein the rate or time duration for theconformational signal change from the conformationally labeledexonuclease is distinguishable for the nucleotide species that areremoved.
 16. The method of claim 14, wherein the conformationallylabeled exonuclease comprises an optical probe.
 17. The method of claim16, wherein the conformational signal change comprises a change inwavelength or intensity.
 18. The method of claim 16, wherein the opticalprobe comprises a fluorophore.
 19. The method of claim 14, wherein theconformationally labeled exonuclease comprises a pair of optical probes.20. The method of claim 19, wherein the conformational signal changecomprises increased FRET, decreased FRET, increased fluorescencequenching or decreased fluorescence quenching.